Dietary food supplement containing natural cyclooxygenase inhibitors and methods for inhibiting pain and inflammation

ABSTRACT

The present invention describes food supplements that contain one or more fruit extracts useful for pain relief and anti-inflammation. The food supplements may be used to inhibit inflammation mediated by cyclooxygenase and more particularly by cyclooxygenase-2.

RELATED APPLICATIONS

The present application is a continuation-in-part of PCT ApplicationSerial No. US00/23423 filed Aug. 25, 2000, which is a continuation ofU.S. Provisional Application Ser. No. 60/151,280 filed Aug. 27, 1999 andU.S. Provisional Application Ser. No. 60/151,278 filed Aug. 27, 1999,all of which are herein incorporated by reference.

BACKGROUND

The present invention relates to dietary food supplements that areuseful for the relief of pain or inflammation, and also for theinhibition of biochemical pathways related to pain or inflammationtransmission. These food supplements contain flavonoids, and moreparticularly, certain anthocyanins.

Today, many consumers seek natural alternatives to syntheticpharmaceutical products to aid with a variety of ailments experiencedduring daily life. Thus, dietary food supplements containing naturalsubstances such as St. Johns wort, gingko biloba, ginseng, and othershave recently been marketed for a variety of purposes. To date, however,it is believed that no product containing natural substances isavailable to provide for the relief of pain and/or inflammationequivalent to non-steroidal anti-inflammatory drugs (“NSAIDs”).

At the present, pain and inflammation are commonly treated by the use ofaspirin, ibuprofen (Motrin®, Advil®), and other similar substancescommonly known as NSAIDs. Inflammation is transmitted, in part, by aclass of compounds known as prostaglandins, which are released by a hostin response to mechanical, thermal, chemical, bacterial, and otherinsults (Moncada et al., Handbook of Exp. Pharm. Vol 50-1, SpringerVerlag, pp 588-616,1978; Samuelsson, Science, 220: 568-575, 1983; Davieset al., Ann. Rev. Immunol. 2:335-357,1984). Prostaglandin synthesis isaccomplished in a stepwise manner by a ubiquitous complex of microsomalenzymes. The first enzyme in this biosynthetic pathway is prostaglandinendoperoxide synthase. This enzyme also is referred to in the art asfatty acid cyclooxygenase. There are two isoforms of this enzyme knownas cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), respectively(Smith, Am. J. Physiol., 268:F181-F191, 1992).

Although substances such as aspirin inhibit prostaglandin production andthus, pain and/or inflammation, they may cause stomach problems and orulcers. To address these problems, drugs have been developed to targetspecific pain pathways in the hope that some of the problems associatedwith aspirin, ibuprofen, and other similar substances will be reduced ifnot completely eliminated. One such drug is Celebrex™, which apparentlytargets a specific pain pathway and thus, does not have some of thedisadvantages associated with substances such as aspirin. In particular,NSAIDs prevent the production of prostaglandins by inhibiting enzymes inthe human arachidonic acid/prostaglandin pathway. Drugs like Celebrex™,however, distinguish between COX-1 and COX-2 and are touted as havingless of the side effects associated with normal NSAIDs.

As noted above, many consumers prefer natural substances to syntheticdrugs. Therefore, it is clear that there is a need for a natural andpharmacologically acceptable composition for use in relieving oralleviating pain, inflammation, and the symptoms associated with theseconditions. In addition, there is a need for a natural composition thatprovides relief from pain and inflammation with minimal side effects tothe gastrointestinal system. The present invention addresses that needby providing a dietary food supplement containing an extract from one ormore anthocyanin-containing plants having a native active fraction thatprovides pain relief, anti-inflammation activity, and/or preferentialCOX-2 inhibition. The supplement contains an amount of the fraction in aproportion by dry weight of other components that significantly exceedsa proportion of the fraction present by dry weight in juice obtainedfrom the plant material. In general, the active fraction includesflavonoids, and in particular, anthocyanins.

Unless otherwise specifically stated, all percentages used in thespecification and claims are weight percentages.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows a flow sheet of one embodiment of a process for obtainingand concentrating desirable anthocyanins from anthocyanin-containingplants.

FIG. 2 is a visual analogue scale rating pain intensity before and aftertaking one or two tablets of a berry blend supplement in accordance withthe present invention and Example 7.

FIG. 3 shows the percentage of pain relief after taking one or twotablets of a berry blend supplement in accordance with the presentinvention and Example 7.

FIG. 4 shows the breakdown of pain episodes by types for the clinicaltrial described in Example 7.

FIG. 5 shows the time to effect pain relief and the overall rating ofone or two tablets of a berry blend supplement in accordance with thepresent invention and Example 7.

FIG. 6 shows the comparative sucrose permeability of ibuprofin and aberry blend supplement in accordance with the present invention andExample 7.

DETAILED DESCRIPTION OF THE INVENTION

Prostaglandins (which include PGE₂, PGD₂, PGF₂, PGI₂ and other relatedcompounds) represent a diverse group of autocrine and paracrine hormonesthat are derived from the metabolism of fatty acids. They belong to afamily of naturally occurring eicosanoids (prostaglandins, thromboxanesand leukotrienes) that are not stored as such in cells, but arebiosynthesized on demand from arachidonic acid, a 20-carbon fatty acidthat is derived from the breakdown of cell-membrane phospholipids. Undernormal circumstances, the eicosanoids are produced at low levels toserve as important mediators of many and diverse cellular functionswhich can be very different in different types of cells. Theprostaglandins, however, also play critical roles in pathophysiology. Inparticular, inflammation is both initiated and maintained, at least inpart, by the overproduction of prostaglandins in injured cells. Thecentral role that prostaglandins play in inflammation is underscored bythe fact that those aspirin-like non-steroidal anti-inflammatory drugs(NSAIDS) that are most effective in the therapy of many pathologicalinflammatory states all act by inhibiting prostaglandin synthesis.Unfortunately, the use of these drugs is often limited by the sideeffects (gastrointestinal bleeding, ulcers, renal failure, and others)that result from the undesirable reduction in prostaglandins in normalcells that now suffer from a lack of those autocrine and paracrinefunctions that are required for the maintenance of normal physiology.The development of new agents that will act more specifically byachieving a reduction in prostaglandins in inflamed cells withoutaltering prostaglandin production in other cells is a goal for futurepain and inflammation therapy.

The cyclooxygenase reaction is the first step in the prostaglandinsynthetic pathway; an enzyme (PGHS) with prostaglandin G/H syntheticactivity converts arachidonic acid into the endoperoxide PGG₂, whichthen breaks down to PGH₂ (the two reactions are carried out by a singleenzyme). PGH₂ is in turn metabolized by one or more prostaglandinsynthase (PGE₂ synthase, PGD₂ synthase etc.) to generate the final“2-series” prostaglandins, PGE₂, PGD₂, PGF₂, PGI₂ and others thatinclude the thromboxanes, TXA₂. The first step (PGHS) is the one that israte-limiting for prostaglandin synthesis. As such, the PGHS-mediatedreaction is the principal target for anti-inflammatory drug action; andit is inhibition of PGHS activity that accounts for the activity of theNSAIDS (aspirin, acetominophen, ibuprofen, naproxen, indomethacin) andothers that limit the overproduction of prostaglandins in inflammation(the desired therapeutic goal) and reduce the normal production ofprostaglandins in uninflamed cells (which produces the undesirable sideeffects).

In addition to the abnormal changes associated with inflammation,multiple other factors are known to influence prostaglandin productionLinder experimental conditions. These include growth factors, cAMP,tumor promoters, src activation and interleukins 1 and 2, all of whichincrease overall cellular PGHS activity. The adrenal glucocorticoidhormones and related synthetic anti-inflammatory steroids also inhibitprostaglandin synthesis, but their metabolic site of action is not welldefined.

The primary and perhaps sole action of most non-steroidalanti-inflammatory agents is to inhibit the enzyme prostaglandin G/Hsynthase, also known as cyclooxygenase, which serves as the firstcommitted step in the biosynthesis of prostaglandins.

It is well established that cyclooxygenase exists in two isoforms, COX-1and COX-2. The constitutively expressed form, COX-1, has beenextensively studied and proposed to be involved in the maintenance ofprostaglandin mediated physiological functions. In contrast, COX-2, theinducible form, is present in negligible amounts under normal conditionsbut is substantially induced in vivo under inflammatory conditions.Clearly, COX-1 and COX-2 serve different physiological and pathologicalfunctions.

The most widely available NSAIDs are non-selective cyclooxygenaseinhibitors, inhibiting both isoforms indiscriminately. When dualinhibitor NSAIDs (NSAIDs that inhibit both the COX-1 and COX-2 enzymes)are used long-term to relieve pain and/or inflammation, the risk ofdeveloping gastrointestinal complications is moderate to high. SelectiveCOX-2 inhibitors have been sought ever since it was discovered that theenzyme has two distinct isoforms. More recently, COX-2 specificinhibitors have been developed but it has been suggested that they toohave side effects. In view of the above, there remains a need for a safeand effective method of treating pain and inflammation with minimalgastrointestinal side effects. The present invention addresses such aneed.

The present invention describes a natural alternative to NSAIDs thatpreferentially inhibits for COX-2 activity and ameliorates inflammationand pain mediated by COX-2 while remaining gentle on the stomach andintestines. The invention shows that extracts or other concentratedforms of certain anthocyanin-containing plants possess ananti-inflammatory activity greater than the anti-inflammatory activityfound in the natural plant. This observation is exploited to provide afood supplement that comprises an extract having an anti-inflammatoryactivity greater than the anti-inflammatory activity found in thenatural plant and a pharmaceutically acceptable diluent or excipient. Inother words, the present invention provides extracts obtained fromanthocyanin-containing plants, particularly fruits, to provide selectiveCOX-2 inhibition.

Alternatively, extracts from anthocyanin-containing plants mayselectively inhibit activity of COX-1. Studies have indicated that someCOX-1 inhibition in a pain and/or anti-inflammatory medication providesa beneficial effect to the cardiovascular system.

Accordingly, the present invention contemplates a food supplement thatcontains at least one anthocyanin derived from an extract of ananthocyanin-containing plant to selectively inhibit activity of COX-1 orCOX-2. As used herein, the term “extract” includes any preparationobtained from plant, fruit, and vegetable using different extractionmethods.

A. Identification of Anthocyanin-Containing Sources

There is a growing need for dietary supplements that contain beneficialphytochemicals that are naturally found in plants. These naturallyoccurring phytochemicals can be classified in several different groups.One of the more important groups is the flavonoids, which in turn, canbe classified into several groups. One important group of flavonoids isthe anthocyanins. Anthocyanins are most prevalent in the flowers andfruits that are red, blue, and intermediate colored such as cherries(sweet, sour (or tart)), acerola cherry, blue plums, bilberry,blackberry, currant, chokeberry, blueberry, strawberry, cranberry,boysenberry, grapes, raspberry, and elderberry.

The anthocyanins may be obtained from any portion of the plant,including, but limited to, the fruit, flower, stem, leaves, root, bark,and seeds. One of skill in the art, however, will understand thatcertain portions of the plant may contain higher natural levels ofanthocyanins and therefore those portions will be used to obtain thedesired anthocyanins. Methods to determine whether and which portions ofa plant contains anthocyanins are known and therefore, not discussedhere.

Examples of suitable anthocyanin-containing plants include fruits,vegetables, flowers and other plants selected from the group consistingof Acer macrophyllum, Acer platanoides, acerola, Ajuga reptans, apple,apricot, Artict bramble, avocado, banana, barberry, barley, Begoniasemperflorens, Bellis perennis, Bletilla striata, bilberry, black beans,black soybeans, black, blue and purple potatoes, blackberry, blueberry,bog whortleberry, boysenberry, buckwheat, cacao, Camellia sinensis,canarygrass, Caucasian blueberry, Chimonanthus praecox, celery, Cerasusavium, cherry, cherry laurel, chicory, chive, chokeberry, Corneliancherry, cornflower, cotoneaster, cowberry, cranberry, crowberry,chrysanthemum, Cynomorium coccineum, Dahlia variabilis, danewort,deerberry, Dendrobium, dwarf dogwood, Echinacea purpea, eggplant,elderberry, fababean, Fatsia japonica, feijoa, fig, garlic, gerbera,ginseng, Globe artichoke, gooseberry, grapes, guava, hawthorn, hibiscusor roselle, Hibiscus Sabdariffa, highbush blueberry, hollyhock,honeysuckle, Ipomoea purpurea, Iris ensata, Java plum, Jerusalemartichoke, kokum, Laeliocattleya spp, lentil, loganberry, lupine,lychee, maize, mango, mangosteen, maqui, Matthiola incana, meconopsis,Metrosideros excelsa, millet, mountain ash berry, mulberry, myrtleberry, olive, onion, orange, ornamental cherry, passion fruit, pea,peach, peanut, pear, perilla, petunia, Phalaenopsis spp. Phalsa,Pharbitis spp. Pineapple, pistachio, plum, pomegranate, Phragmitesaustralis, purple carrot, quince, rabbiteye blueberry, radish, red andblack currant, red and black raspberry, red cabbage, rice, rhubarb,rosehip, rye, saffron, sarracenia, sheepberry, Sophronitis coccinea,sorghum, sparkleberry, strawberry, Fragaria Vesca, sugarcane, sunflower,sweet cherry, sweet potato, tamarillo, tamarind, taro, tart cherry,Tulip greigii, turnip, water lily, Weigela spp, wheat, wild rice,Verbena hybrida, yam and mixtures thereof.

The chemistry of anthocyanins is based on 2-phenylbenzopyrylium(flavylium) having the following structure:

If this basic formula is substituted with hydroxy or methoxy groups at2, 3, 4, 5, 7, 3′ or 5′, the resultant compounds are known asanthocyanidins, which are water insoluble, unstable to light and rapidlydestroyed by alkali and thus not found too often in plants. Table 1below shows the structure for several of the anthocyanidins: TABLE 1Substitution Pattern Name 3 5 6 7 3′ 4′ 5′ Color Cyanidin OH OH H OH OHOH H Orange (Cy) red Delphinidin OH OH H OH OH OH OH Bluish-red (Dp)Malvidin OH OH H OH OME OME OME Bluish-red (Mv) Pelargonidin OH OH H OHH OH H Orange (Pg) Peonidin OH OH H OH OME OH H Orange- (Pn) redPetunidin OH OH H OH OME OH OH Bluish-red (Pt)

Anthocyanins are the glycosides of the above compounds and are morestable and found as native substances in the leaves, flowers and fruitsof plants. The anthocyanins may be hydrolyzed to produce anthocyanidins(the aglycone form) and sugars.

The total number of anthocyanins found in nature is extremely large,since many mono, di and tri-saccharides may be glycosylated at the 3, 5or 7 positions and also since the sugar at position 3 may be acylated(often with p-coumaric acid). Thus, a particular fruit may have 20 ormore anthocyanins including the 3,5-diglucosides, the 3-mono-glucoside,the 3-(6-O-p-coumaryl-glucoside)-5-glucosides and the3-(6-O-p-coumaryl-glucoside) of cyanidin, delphinidin, petunidin,pelargonidin and malvidin. The color of anthocyanins is determined bytheir molecular structure and the physiochemical nature of the medium inwhich they are present.

In accordance with the present invention, the extract contains one ormore anthocyanins and anthocyanidins selected from the group consistingof peonidin, cyanidin, pelargonidin, delphinidin, petunidin, malvidin,apigenindin, auratinidin, capensinidin, europinidin, hirsutidin,6-hydroxycyanidin, luteolinidin, 5-methylcyanidin, pulchellidin,rosinidin, tricetinidin, derivatives and mixtures thereof. In oneembodiment, the anthocyanins and anthocyanidins are selected from thegroup consisting of cyanidin, peonidin, malvidin, petunidin,delphinidin, their glycoside derivatives, and mixtures thereof. In yetanother embodiment, the extract contains at least one cyanidin-basedanthocyanin.

Anthocyanins that may be useful in the inventions described hereininclude, but are not limited to, cyanidin-3-glucoside; cyanidin3-glucosylrutinoside; cyanidin-3-gentibioside; cyanidin-3-rutinoside,cyanidin-3-sambunigrin, cyanidin-3-samb-5-glucoside,cyanidin-3-galactoside, peonidin-3-rutinoside, peonidin-3-glucoside,peonidin-3-galactoside, peonidin, cyanidin, cyanidin-3 sophoroside,pelargonidin, delphinidin, delphinidin-3-glucoside,delphinidin-3-galactoside, petunidin, petunidin-3-glucoside,petunidin-3-galactoside, malvidin, malvidin-3-arabinoside,malvidin-3-glucoside, malvidin-3-galactoside, kaempferol, hesperidin,gentiodelphin, platyconin, cinerarin and the like.

It has been found that hydrolyzed anthocyanins (i.e. anthocyanidins) mayprovide greater COX inhibition activity as compared to the anthocyaninand its glycoside derivatives. It is believed that the anthocyaninsprovide little, if any, COX inhibition, particularly COX-1 inhibition.However, it has also been found that it may be advantageous to includeanthocyanins in the dietary food supplement and allow them to behydrolyzed in vivo to the anthocyanidin form. It is believed that theanthocyanins can be absorbed or passed through the gastrointestinaltract without inhibiting the COX 1 enzyme there. As a result, the amountof prostaglandins generated by the COX 1 enzyme on the gastrointestinaltract remains normal or high enough to maintain the GI lining. After theabsorption, the sugar moieties are cut off and the active form, theanthocyanidins, are transported to the sites where the COX 2 enzymes arebeing induced, thereby inhibiting the COX 2 enzymes and providing relieffrom pain and inflammation. As a result, the present invention isbelieved to provide an advantage over currently available COXinhibitors, such as NSAIDs, since there will be little inhibition of theCOX-1 in the gastrointestinal tract, with a possible reduction in sideeffects.

As described further in the Examples, cyanidin may provide a greateramount of COX inhibition as compared to the other anthocyanidins.Accordingly, it may be desirable to provide a dietary supplement havinga high concentration of cyanidin-based anthocyanins to achieve pain andinflammation relief. It has further been found that anthocyaninscontaining only a single sugar moiety may provide a greater amount ofCOX inhibition as compared to di-saccharchide, tri-saccharide, or othermultiple saccharide anthocyanins. Table 2 identifies the cyanidin-based,monosaccharide anthocyanins found in many natural sources. TABLE 2Cyanidin-based Commercial anthocyanins found source of an in thissource¹ Weight Anthocyanin- extract or other percentage, if provided,Cyanidin-based containing Latin concentrated is relative to all knownmono-saccharides COX1/COX2 plant Name product anthocyanins in thissource found in this source Ratio² Acer N/A Cyanidin derivative N/A N/Amacrophyllum Acer platanoides N/A Cyanidin 3-(2″,3″- N/A N/Adigalloyl-beta- glucopyranose (3%) Cyanidin 3-(2″-galloyl-beta-glucopyranose (37%) Cyanidin 3-beta- glucopyranoside (60%) AcerolaMalpighia N/A Cyanidin-3-glucoside Cyanidin-3- N/A marginata glucosideAjuga reptans N/A Cyanidin 3-(di-p- N/A N/A coumaroyl) sophoroside-5-glucoside Apple Malus spp N/A Cyanidin 3-galactoside Cyanidin 3- N/ACyanidin 3-arabinoside galactoside Cyanidin 3-glucoside Cyanidin 3-Cyanidin 3-xyloside arabinoside Cyanidin 3- glucoside Cyanidin3-xyloside Apricot Prunus armeniaca N/A Cyanidin-3-glucoside Cyanidin-3-N/A glucoside Artic bramble Rubus spp N/A N/A N/A N/A Avocado Persea sppN/A Acylated cyanidin 3,5- Cyanidin 3- N/A diglucoside galactosideCyanidin 3-galactoside Banana Musa acuminata N/A N/A N/A N/A M.balbisiana Barberry Berberis spp. N/A Cyanidin-glucosideCyanidin-glucoside N/A Barley Hordeum vulgare N/A Cyanidin and cyanidinN/A N/A glycosides Bean Phaseolus N/A Cyanidin 3-glucoside Cyanidin 3-N/A vulgaris (several Cyanidin 3,5-diglucoside glucoside cultivars)Begonia N/A Cyanidin derivative N/A N/A semperflorens cvs Benibana-chaCamellia sinensis N/A Cyanidin 3-O-beta-D- Cyanidin 3-O-beta- N/Agalactoside D-galactoside Bellis perennis N/A 3 Cyanidin 3-derivativesN/A N/A Bletilla striata N/A Acylated cyanidin 3,7,3′- N/A N/Atriglucoside derivatives Bilberry Vaccinium Artemis/Iprona;Cyanidin-3-galactoside Cyanidin-3- >1.3 myrtillus Indena (22%);galactoside Cyanidin-3-glucoside Cyanidin-3- (9%); glucoside Black beansPhaseolus N/A Cyanidin-3-glucoside Cyanidin-3- N/A (96%) glucosideBlackberry Moriferi veri N/A Cyanidin-glucoside (70-100%)Cyanidin-glucoside N/A (European and Rubus caesius Cyanidin-rutinosideAmerican) R. alleghniensis, R. argutus, R. cuneifolius, R. setosus, R.trivialis. Black grapes Many varieties N/A N/A N/A N/A Black potatoesSolanum N/A Cyanidin-glycosides* N/A tuberosum Black raspberry Rubus N/ACyanidin-sambubioside Cyanidin- >1.3 occidentalis (20%); sambubiosideCyanidin- Cyanidin-glucoside xylosylrutinoside (40%); Cyanidin-glucoside(17%); Cyanidin-rutinoside (23%) Black soybeans Glycine max N/ACyanidin-3-glucoside Cyanidin-3- N/A (96%) glucoside Blueberries Fivecommon N/A Cyanidin-glucoside (3%); Cyanidin-glucoside N/A Vaccinium sppCyanidin-galactoside Cyanidin- (3%); galactoside Cyanidin-arabinosideCyanidin-3- (3%) arabinoside Bog Vaccinium N/A Cyanidin 3-glucosideCyanidin 3- whortleberry uliginosum (14%) glucoside (14%) Cyanidin3-arabinoside Cyanidin 3- (10%) arabinoside (10%) Cyanidin 3-galactosideCyanidin 3- (6.5%) galactoside (6.5%) Boysenberry new ZealandCyanidin-3-sophoroside Cyanidin-3- N/A (44.5%) glucosideCyanidin-3-glucoside (26.4%); Cyanidin-3- glycosylrutinoside (25.8%);Cyanidin-rutinoside (3.3%) Buckwheat Fagopyrum N/A Cyanidin 3-glucosideCyanidin 3- N/A species Cyanidin 3-galactoside glucoside Cyanidin 3-galactoside Cacao Theobroma cacao N/A Cyanidin 3-glucoside Cyanidin 3-N/A (suspected) glucoside (suspected) Celery Apium spp N/A N/A N/A N/ACherry laurel Prunus N/A Cyanidin 3-arabinoside Cyanidin 3- N/Alaurocerasus arabinoside Chicory Cichorium intybus N/A Cyanidin3-glucoside Cyanidin 3-glucoside N/A Chive Allium N/A Cyanidin3-glucoside Cyanidin 3- N/A schoenoprasum Cyanidin 3- glucosideacetylglucoside Cyanidin 3-(6- malonylglucoside) Cyanidin 3-(3,6-dimalonylglucoside) Chokeberry Aronia Artemis/IpronaCyanidin-3-galactoside Cyanidin-3- >1.3 melanocarpa (64.5%); galactosideCyanidin-3-arabinoside Cyanidin-3- (28.9%); arabinosideCyanidin-3-glucoside Cyanidin-3- (2.4%); glucoside Cyanidin-3-xylosideCyanidin-3-xyloside (4.2%) Coffee Coffea arabica N/A Cyanadin3-glycoside Cyanadin 3- N/A cv. Bourbon Cyanadin 3,5- glycoside Vermelhodiglycoside Cotoneaster Cotoneaster N/A Cyanidin 3-glucoside Cyanidin 3-N/A Medic. Spp.) Cyanidin 3-galactoside glucoside Cyanidin 3-rutinosideCyanidin 3- galactoside Cowberry or V. vitis-idaea N/A Cyanidin3-galactoside Cyanidin 3- N/A Lingonberry Cyanidin 3-arabinosidegalactoside Cyanidin 3-glucoside Cyanidin 3- arabinoside Cyanidin 3-glucoside Chimonanthus N/A Cyanidin 3-O-glucoside Cyanidin 3-O- N/Apraecox Acylated cyanidin 3-O- glucoside glucoside Cyanidin glycosideCranberry Vaccinium Ocean Spray Cyanidin-galactoside Cyanidin- N/A(American and macrocarpon (16-24%) galactoside European) V. oxycoccusCyanidin-arabinoside Cyanidin- (13-25%) arabinoside Crowberry Empetrumnigrum N/A Cyanidin 3-glucoside Cyanidin 3- N/A Cyanidin 3,5-diglucosideglucoside Cyanidin 3-rutinoside Cyanidin 3-sophoroside ChrysanthemumDendranthema N/A Cyanidin 3-O-(6′-O- N/A N/A Grandiflorum malonyl-beta-glucopyranoside Currant (red Ribes rubrum N/A Cyanidin-glucoside(2-10%), Cyanidin-glucoside, N/A and black) R. nigrumCyanidin-rutinoside (8-17%), Cyanidin- Cyanidin-sambubiosidesambubioside (9-31%), Cyanidin-sophoroside (4-9%), Cyanidin-xylosylrutinoside (28-73%), Cyanidin- glucosylrutinoside (14-28%),Cynomorium N/A Cyanidin 3-O-glucoside Cyanidin 3-O- N/A coccineum (92%)glucoside (92%) Cyanadin 3-O-(6-O- rhamnosylglucoside (8%) DanewortSambucus ebulus N/A Cyanidin 3- Cyanidin 3- N/A xylosylglucosidesambubioside Cyanidin 3- Cyanidin 3- sambubioside glucoside Cyanidin 3-sambubioside-5- glucoside Cyanidin 3,5 diglucoside Cyanidin 3-glucosideCyanidin 3- arabinoglucoside Dendrobium Phalaenopsis spp N/A Cyanidinderivatives N/A N/A Dwarf dogwood Cornus suecica N/A Cyanidin3-glucoside Cyanidin 3- N/A (4%) glucoside (4%) Cyanidin 3-galactoside(16%) 2 Cyanidin derivatives (80%) Echinacea Echinacea spp. N/A N/A N/AN/A Elderberry Sambucus Artemis/Iprona Cyanidin-3-glucosideCyanidin-3- >1.3 nigra (42%) glucoside Cyanidin-3- sambubioside (43%)Cyanidin-3,5-diglucoside (2%) Cyanidin-3- sambubioside —5- glucoside(9%) Gentiana spp N/A Cyanidin 3-O-beta-D- Cyanidin 3-O-beta- N/Aglucoside and 3 other D-glucoside derivatives Fatsia japonica N/ACyanidin 3-lathyroside N/A N/A Feijoa Feijoa sellowiana N/A Cyanidin3-glucoside Cyanidin 3- N/A glucoside Fig Ficus carica spp N/A Cyanidin3- Cyanidin 3- N/A rhamnoglucoside glucoside Cyanidin 3,5-diglucosideCyanidin 3-glucoside Forsythia X N/A Cyanidin derivatives N/A N/Aintermedia cv Spring Glory Garlic Allium sativum N/A Cyanidin3-glucoside Cyanidin 3- N/A Cyanidin 3-glucoside glucoside monoacylatedCyanidin 3-glucoside triacylated Ginseng Panax ginseng N/A Cyanidin3-O-β-D- N/A N/A Panax xylopyranyl-(1-2)-β-D- quinquefoliusglucopyranoside Globe artichoke Cynara scolymus N/A Cyanidin 3- N/A N/Acaffeylglucoside Cyanidin 3- caffeyIsophoroside Cyanidin 3-dicaffeyIsophoroside Gooseberry Ribes spp N/A Cyanidin 3-glucosideCyanidin 3- N/A Cyanidin 3-rutinoside glucoside Grape Vinis vinifera N/ACyanidin 3- Cyanidin 3- N/A monoglucoside monoglucoside Cyanidin 3-monoglucoside-acetate Cyanidin 3- monglucoside-p- coumarate GuavaPsidium guajavica N/A Cyanidin 3-glucoside Cyanidin 3- N/A glucosideHawthorn Crataegus spp N/A Cyanidin 3-galactoside Cyanidin 3- N/ACyanidin 3-arabinoside galactoside Cyanidin 3-glucoside Cyanidin 3-glucoside Hibiscus or Hibiscus N/A Cyanidin-sambubioside Cyanidin- N/ARoselle sabdariffa (30%) sambubioside Hollyhock Althaea rosea N/ACyanidin 3-glucoside Cyanidin 3- N/A Cyanidin 3-rutinoside glucosideOther cyanidin glucosides Honeysuckle Lonicera nitida N/A Cyanidin3-rutinoside Cyanidin 3- N/A Cyanidin 3-glucoside glucoside JapaneseIris ensata N/A Cyanidin 3RG N/A N/A garden iris Cyanidin 3RG5G Cyanidin3Rgac5G Ipomoea N/A Six acylated cyanidin 3- N/A N/A purpureasophoroside-5- glucosides Java plum Myrciaria N/A Cyanidin 3-glucosideCyanidin 3- N/A jaboticaba glucoside Jerusalem Helianthus N/A N/A N/AN/A artichoke tuberosus Kokum Garcinia indica N/A Cyanidin 3-glucosideCyanidin 3- N/A Cyanidin 3- glucoside sambubioside Cyanidin 3-sambubioside Laeliocattleya cv N/A Acylated cyanidin N/A N/A Mini purplederivatives Lactuca sativa N/A Cyanidin 3-O-(6″- N/A N/Amalonylglucoside) Loganberry Rubus N/A Cyanidin-sophorosideCyanidin-glucoside N/A loganbaccus (48.1%), Cyanidin-glucoside (21.6%),Cyanidin-rutinoside (6.2%) Lupine Lupinus spp N/A Cyanidin glycosidesN/A N/A presence confirmed Lychee Litchi chinensis N/A Cyanidin3-glucoside Cyanidin 3- N/A Cyanidin 3-galactoside glucoside Cyanidin3-rutinoside Cyanidin 3- galactoside Maize Zea mays N/A Cyanidin3-glucoside Cyanidin 3- N/A Cyanidin 3-(6″- glucoside malonylglucoside)Cyanidin 3- (3″,6″dimalonyl- glucoside) Mango Mangifera indica N/ACyanidin glycosides N/A N/A Mangosteen Garcina N/A Cyanidin3-sophoroside Cyanidin 3- N/A mangostana Cyanidin 3-glucoside glucosideMaqui Aristotelia N/A Cyanidin 3-,5- N/A N/A chilensis diglucosideMatthiola incana N/A Four acylated cyanidin N/A N/A 3-sambubioside-5-glucosides Millet Pernnisetum N/A Cyanidin 3-glucoside Cyanidin 3- N/Aamericanum glucoside Mountain ash Sorbus spp N/A Cyanidin 3-galactosidN/A N/A berry Cyanidin 3,5-diglucoside Cyanidin 3-β-D- glucopyranosideMulberry Morus nigra N/A Cyanidin 3-glucoside Cyanidin 3- N/A Cyanidin3,5-diglucoside glucoside Cyanidin 3-rutinoside Cyanidin 3-sophorosideMyrtle berry Myrtus communis N/A Cyanidin 3-glucosides Cyanidin 3- N/ACyanidin 3,5- glucosides diglucosides Olive Olea europaea N/A Cyanidin3-rutinoside Cyanidin 3- N/A Cyanidin 3-glucoside glucoside Cyanidinderivatives Onion Allium sepa N/A Cyanidin 3-glucoside Cyanidin 3- N/ACyanidin 3-diglucoside glucoside Cyanidin 3-laminarioside Orange Citrussinensis N/A Cyanidin 3- Cyanidin 3- N/A glucoside(95%) glucosideCyanidin 3,5-diglucoside Passion fruit Pasiflora edulis N/A Cyanidin3-glucoside Cyanidin 3- N/A glucoside Pea Pisum sativum N/A Cyanidin 3-N/A N/A sophoroside-5- glucosides Cyanidin 3- sambubioside-5- glucosidesPeach Prunus persica N/A Cyanidin 3-glucoside Cyanidin 3- N/A Cyanidin3-rutinoside glucoside Cyanidin derivatives Peanut Arachis hypogaea N/ACyanidin glucosides N/A N/A Pear Pyrus communis N/A Cyanidin3-galactoside Cyanidin 3- N/A Cyanidin 3-arabinoside galactosideCyanidin 3- arabinoside Perilla Perilla frutescens N/A Cyanidin3,5-diglucoside N/A N/A Cyanidin 3,5-derivatives Petunia spp N/ACyanidin 3-rutinoside N/A N/A Phalsa Grewia spp N/A Cyanidin 3-glucosideCyanidin 3- N/A glucoside Pineapple Ananas comosus N/A Cyanidin3-galactoside Cyanidin 3- N/A galactoside Pistachio Pistacia vera N/AN/A N/A N/A Pragmites N/A Cyanidin-3 derivatives N/A N/A australis Plum2000 varieties N/A Cyanidin-glucoside Cyandin-glucoside N/A 15 species(37%) Cyanidin-rutinoside (45%) Pomegranate Punica granatum N/ACyanidin-glucoside (30% Cyanidin-glucoside N/A Cyanidin-diglucoside(17%) Purple carrot Daucus carota N/A Cyanidin-glucosideCyanidin-glucoside N/A Cyanidin- Cyanidin- glucosylgalactosidegalactoside Cyanidin-digalactoside Cyanidin-galactoside Quince Cydoniaoblonga N/A Cyanidin 3-glucoside Cyanidin 3- N/A Cyanidin3,5-diglucoside glucoside Cyanidin derivatives Radish Raphanus sativusN/A Acylated cyanidin 3- N/A N/A sophoroside-5-glucoside Acylatedcyanidin 3- diglucoside-5-glucoside Red cabbage Brassica oleracea N/ACyanidin-glycosides* N/A N/A var. capitata Reed Phalaris N/A Cyanidin3-glucoside Cyanidin 3- N/A canarygrass arundinacea Cyanidin 3-(6″-glucoside malonylglucoside) Cyanidin 3- (3″,6″dimalonyl- glucoside) Redonion Allium cepa N/A Cyanidin 3-glucoside Cyanidin 3- N/A Acylatedcyanidin 3- glucoside glucoside derivatives Red petunia Petunia spp N/ACyanidin 3-glucoside Cyanidin 3- N/A Cyanidin 3-sophoroside glucosideRed raspberry Rubus idaeus N/A Cyanidin-glucoside Cyanidin-glucoside N/A(17%); Cyanidin-rutinoside (7%); Cyanidin-sophoroside (50%); Cyanidin-glycosylrutinoside (26%); Cyanidin-diglucoside Rhubarb Rheum spp N/ACyanidin 3-glucoside Cyanidin 3- N/A Cyanidin 3-rutinoside glucosideRice Oryza spp N/A Cyanidin 3-glucoside Cyanidin 3- N/A Cyanidin3-rhamnoside glucoside Cyanidin 3,5-diglucoside Rosehip Rosa canina N/ACyanidin 3-rutinoside Cyanidin 3- N/A Cyanidin 3-glucoside glucosideCyanidin 3,5-diglucoside Rye Secale cereale N/A Cyanidin 3-glucosideCyanidin 3- N/A Cyanidin 3- glucoside rhamnosylglucoside Cyanidin 3-rhamnosyldiglucoside Cyanidin 3-rutinoside Cyanidin 3-rutinosidederivatives Cyanidin 3-gentiobioside Sheepberry Viburnum spp N/ACyanidin 3-glucoside Cyanidin 3- N/A Cyanidin 3- glucosidearabinosylsambubioside Sorghum Sorghum bicolor N/A Cyanidin N/A N/ACyanidin glycosides Sparkleberry V. arboreum N/A Cyanidin 3-glucosideCyanidin 3- N/A Cyanidin 3-arabinoside glucoside Cyanidin 3-galactosideStrawberry Fragaria N/A Cyanidin- Cyanidin-glucoside N/A ananassaglucoside(minor) Sunflower Helianthus N/A Cyanidin 3-glucoside Cyanidin3- N/A annuus Acylated cyanidin 3- glucoside glucoside Cyanidin3-xyloside Cyanidin 3-xyloside Acylated cyanidin 3- xyloside Cyanidin3-vanillyl- sambubioside Sweet cherry Prunus avium N/ACyanidin-glucoside Cyanidin-glucoside N/A Cyanidin-rutinoside;Cyanidin-3-sophoroside Sweet potato Ipomoea batatas N/A Cyanidinderivatives N/A N/A Sophronitis N/A Five acylated cyanidin N/A N/Acoccinea 3,3′,7-triglucosides. Tamarillo or Cyphomandrea N/A Cyanidin3-rutinoside Cyanidin 3- N/A tomato tree betacea Cyanidin 3-glucosideglucoside Tamarind Tamarindus N/A Cyanidin 3-glucoside Cyanidin 3- N/Aindica glucoside Taro Colocasia N/A Cyanidin 3-glucoside Cyanidin 3- N/Aesculenta Cyanidin 3-rutinoside glucoside Tart Cherry Prunus cerasusNutrilite Cyanidin-3-rutinoside- N/A >1.3 (balaton) cv. Balaton hexose(75%); Cyanidin-3-rutinoside- pentose (3%); Cyanidin-3-rutinoside (18%);Tart cherry Prunus cerasus Nutrilite Cyanidin-3-sophorosideCyanidin-3- >1.3 (montmorency) cv. Montmorency (80%); glucoside (20%)Cyanidin-3-glucoside (20%) Tulip Tulipa spp N/A Cyanidin 3-O-(6″- N/AN/A rhamnosylglucosides Cyanidin 3-O-derivative Turnip Brassica rapa N/ACyanidin 3-glucoside Cyanidin 3- N/A Cyanidin 3-diglucoside- glucoside5-glucoside Cyanidin 3,5-diglucoside Water lily Nymphaea alba N/ACyanidin 3-O-(6″-acetyl- Cyanidin 3-O- N/A beta-galactopyrosinasegalactoside (2%) (23%) Cyanidin 3-O- galactoside (2%) Weigela spp N/ACyanidin 3-O-glucoside Cyanidin 3-O- N/A Cyanidin 3-O-glucose- glucosidexylose Wheat Triticum spp N/A Cyanidin 3-glucoside Cyanidin 3- N/AAcylated cyanidin glucoside glucoside Cyanidin 3-rutinoside Acylatedcyanidin 3- rutinoside Cyanidin 3-gentiobioside Wild rice Zizaniaaquatica N/A Cyanidin 3-glucoside Cyanidin 3- N/A Cyanidin 3- glucosiderhamnoglucoside Yam Dioscoracea spp N/A Cyanidin 3,5-diglucosideCyanidin 3- N/A Cyanidin 3-glucoside glucoside Cyanidin 3-rhamnoglucoside Cyanidin 3-gentiobioside Acylated cyanidin glucosidesB. Methods for Extracting Anthocyanins

There are various methods for the extraction of anthocyanins known tothose of skill in the art. Some of these methods are described in, forexample, U.S. Pat. No. 5,817,354; U.S. Pat. No. 5,200,186; U.S. Pat. No.5,912,363; U.S. Pat. No. No. 4,211,577; U.S. Pat. No. 4,302,200 (eachincorporated herein by reference).

U.S. Pat. No. 5,817,354 describes a process for removing flavonoids fromcitrus products that cause the bitter taste. The process includescontacting a fluid containing one or more these bitter flavonoids with apolystyrene divinylbenzene resin to bind the flavonoids to the resin.Generally, a centrifugation or ultrafiltration step is used beforecontacting with the polystyrene divinylbenzene resin. The flavonoids canthen be collected by eluting from the resin. While this patent does notdescribe how the flavonoids can be eluted (removed) from the resin,Chandra, et al. (J. Agric. Food Chem., 1062-64, Vol. 41, No. 7 (1993))describe the use of ethanol to elute the anthocyanins. The elutedsolution is then vacuum dried to remove the ethanol.

U.S. Pat. No. 5,912,363 describes a method for the extraction ofproanthocyanidins from plant material. The method involves heating anaqueous mixture of solid plant material, optionally under increasedpressure and reduced oxygen followed by various separation, filtrationand adsorption steps, and the elution of adsorbed proanthocyanidins withpolar solvent. This method also is amenable to reconstituting andrecycling the polar solvent into the elution phase of the method,resulting in decreased solvent consumption and a more cost-effectiveprocess.

U.S. Pat. No. 4,211,577 describes the extraction of plant anthocyanincolors by treating impure materials to insure discrete monomericanthocyanin molecules in solution and then passing the solution throughultrafiltration membranes to retain soluble and/or cloudymacromolecular, e.g., colloidal, impurities upstream that produce, anaging, haze and sediments, and passing the monomeric anthocyaninsdownstream for further concentration as liquid or powder to give astable color concentrate that can be used as a color additive. In thismanner, fruit solids may be treated with sulfur dioxide solutions toionize, decolor and insure the monomeric state of the pigment molecules(change from anthocyanins to chromon 2- and 4-sulfonates).Ultrafiltering the solution to pass the anthocyanins downstream whileretaining upstream the macromolecular components such as pectins,tannins, proteins, complexes thereof, etc. Optionally stripping of thesulfur dioxide from the ultrafiltered solution regenerates the originalanthocyanins from the chromen sulfonates. The anthocyanins can then beconcentrated by evaporation to a highly concentrated liquid from whichunstable pigments with acyl groups in the molecule may optionally beremoved by controlled precipitation at reduced temperatures.

U.S. Pat. No. 4,302,200 describes a process for the extraction ofanthocyanins from a natural product by bringing the natural productcontaining the anthocyanin into contact with a sulfite ion-containingaqueous solution at a temperature of 85° C. or higher for 30 minutes orless, at which time the sulfite ion content of the aqueous solutionfirstly contacting the natural product is adjusted to at least 10,000ppm in terms of SO₂.

U.S. Pat. No. 3,963,700 describes a method of recovering anthocyaninsfrom plant materials such as grape wastes using a tartaric acid-alkanolextraction followed by controlled precipitation of excess tartaric acidas potassium hydrogen tartrate. This patent further describes the use ofthese anthocyanins in the preparation of an artificial grape drinkcolored with the anthocyanin extract.

While it is contemplated that the methods described in the above patentswill be useful in generating the anthocyanins for the anti-inflammatoryproperties described herein, the inventors have developed another methodof extraction of anthocyanins from a natural source.

The method is directed to concentrating flavonoids from plants withoutthe use of undesirable chemicals. The process includes passing asolution containing one or more flavonoids through an ultrafiltrationmembrane to provide a supernatant and a retentate. The supernatant isthen passed through a reverse osmosis membrane to provide a retentateand a permeate, and then the reverse osmosis retentate is collected.

The molecular weight cutoff of the ultrafiltration membrane ispreferably in the range of about 100,000 to about 1,000,000. Themolecular weight cutoff of the reverse osmosis membrane is preferably inthe range from about 1,000 to about 10,000.

The collected retentate contains the anti-inflammatory, COX-2 inhibitingproperties that are described in the present invention. In preferredembodiments the retentate may thus be dried and combined with one ormore excipients to provide a dietary supplement.

Referring now to FIG. 1, there is shown a flow sheet of one embodimentof the process according to the present invention. In accordance withthis embodiment, a plant source 1, particularly a fruit containingflavonoids and more particularly a fruit containing anthocyanincompounds is processed by an extraction method 10 to obtain an extractor juice 2. For example, the plant may be subjected to a juicingoperation or a pressing operation such as a screw or bag press to obtaina cake and a juice. Alternatively, the raw plant may be ground,pulverized or subjected to process to increase the surface area of theplant to facilitate the extraction and separation of the desiredflavonoid compounds from the bulk solids.

To aid in this separation and to obtain a better ultimate recovery ofthe desired anthocyanin compounds, it may be desirable to contact theplant with an extractant 3 to obtain an extract juice) rich inflavonoids (particularly the anthocyanin compounds) and to form a bulksolid residue or cake 4. Preferably, the extractant is water in order tominimize further separation processing steps. The extracting step may bedone using conventional extraction equipment, in countercurrent fashion,batch, or multiple batch extraction.

In addition, the cake may likewise be subjected to an extraction processto increase the recovery of the desired anthocyanin compounds. If thisextraction process step is conducted, it may be desirable to combine theextract from this step with the juice and/or extract (juice) from theprevious step.

The juice may separated from the cake in any known manner using bulkseparation apparatus such as a centrifuge, screen, press, or filter.Prior to ultrafiltration, the bulk solids are desirably separated fromthe liquid by any known bulk separation apparatus. For example, thefollowing may be used a centrifuge, filter, screen, press, etc.

Thereafter, an ultrafiltration process 20 is used to remove suspendedparticles and colloidal high molecular weight components having amolecular weight greater than about 200,000 Daltons. The ultrafiltrationmembrane can be a tubular type, a capillary type, spiral type, hollowfiber, or other suitable type. The membrane can be polysulphone,polyacrylonitrile, polyethersulphone, PVDF or other suitable material.Preferably, the ultrafiltration is conducted using cross-flow. Themolecular weight cut off of the membrane can range from about 20,000Daltons to about 300,000 Daltons, preferably about 200,000 Daltons. Ifthere is no filtration before the ultrafilter, it is preferred to use ahigher molecular weight cut off membrane so that an acceptablefiltration rate can be achieved. Thus, it is contemplated to incorporatea microfiltration step before the ultrafiltration step.

For example, a microfilter may be used to remove suspended particleshaving a size in the range from about 0.01 to about 1 micrometer.

The ultrafiltration can be conducted under a pressure of about 5 toabout 25 bar and at a temperature of about 22° C. to about 80° C. Thisstep primarily removes the lipids, proteins and like colloids, cellfragments, starch, etc. with the advantage that the following RO stepcan be carried out free of the contamination of the membrane(s) thatwould otherwise lead to a reduced filtration rate.

The ultrafiltration step results in a permeate 5 rich in anthocyanincompounds and a retentate 7 containing undesirable compounds. Toincrease the ultimate recovery of the flavonoids and desired anthocyanincompounds, a difiltrate 6 may be provided to the ultrafiltrationmembrane.

The ultrafiltration permeate 5 is subjected to reverse osmosis 30 toprovide a retentate 8 rich in flavonoids, including the anthocyanincompounds, and a permeate 10, which is substantially free of theflavonoids, including the anthocyanin compounds. To increase theultimate recovery of the flavonoids and desired anthocyanin compounds, adifiltrate 9 may be provided to the ultrafiltration membrane. Themembrane to be used for the RO of the present invention can bepolyethersulphone, polysulphone, cellulose acetate, or a polyamide film.

The reverse osmosis can be conducted at a pressure from about 30 toabout 70 bar and at a temperature from about 30° C. to about 80° C.,preferably the temperature is maintained in the range from about 30° C.to about 45° C. In general, the reverse osmosis membrane has a molecularweight cutoff in the range from about 1,000 to about 10,000, preferablyabout 2,000 to provide a retentate.

The retentate contains a higher concentration of the desired anthocyanincompounds than found in the starting plant material. The retentate maybe left in the form of a solution but also may be further concentratedby drying 40 to remove some of the water or may be completely dried toform a powder 11.

Where a more concentrated solution is desired, some of the water may beremoved by conventional means including use of reverse osmosis membraneshaving greater than 90% NaCl retention.

Spray drying is the preferred drying means but other drying methods,e.g. flash drying, freeze drying, fluidized bed drying, ring drying,micron drying, tray drying, vacuum drying, radio-frequency drying, ormicrowave drying, may also be adapted for use in this drying step.Before drying, it may be desirable to add one or more flow controlagents such as maltodextrin (e.g. M100), magnesium hydroxide or otherknown flow controls agents or carriers. In general, it may be desirableto add a flow control agent in an amount from about 20 to 60% by weightof the solid content in the retentate.

When spray drying is used, the total solids content of the retentateshould be at least about 1%, based on the total slurry weight althoughhigher total solids content in the range of at least about 20% to about35% solids would be desired. The higher solids content levels aredesirable since the amount of water that must be removed during thedrying step is accordingly reduced. Consequently, the solids content ofthe retentate will be as high as can be achieved and yet allow efficientprocessing conditions. The upper limit on solids content in theretentate is typically determined by the operating constraints of themembrane used in the reverse osmosis/nanofiltration step as well as thedrying apparatus used.

The temperature of the retentate is not critical. Ambient temperatures,of from about 10-25° C., will generally be preferred. Higher slurrytemperatures may be used, and these may be desirable with certain typesof drying equipment.

Conventional spray drying equipment may be used, and operatingprocedures that are familiar to those experienced in the spray dryingart are applicable to the spray-drying step of this process. Drier(drier gas) outlet temperature is ordinarily used to control theresidual moisture level obtained in the resulting powder. In a spraydrying process, drier outlet temperatures are ordinarily in the range ofabout 40-100° C. In general, it is desirable to maintain the outlettemperature to less than about 80° C. to minimize the potential fordegradation of the desired anthocyanin compounds. It is understood thatthe corresponding drier inlet temperatures are higher, ordinarily in therange of about 90° C. to about 200° C., but preferably less than about150° C.

The product recovered from the drying operation is a free-flowingparticulate solid that typically has a fine granular powder appearanceand is suitable for use as a dietary or food supplement. In this regard,the resulting powder containing the desired one or more anthocyanincompounds is useful as a food or dietary supplement.

The reverse osmosis permeate may be further processed by, for example, aconcentrator 50 to provide a concentrate 12 that may be used to preparea fruit drink.

Of course the above is only one method and it should be understood thatany method, which provide fruit extracts possessing an anti-inflammatoryactivity greater than the anti-inflammatory activity found in thenatural fruit, will be useful in the context of the present invention.

Although any of the above methods are suitable for obtaining the desiredanthocyanin, it is also contemplated that commercially availableextracts may be used for some or all of the requirements of the productsof the present invention. As an example, it is known that ArtemisInternational of Fort Wayne, Ind. supplies juice concentrates andpowders that contain anthocyanins and other flavonoids. Where commercialproducts are used, it is preferred that the anthocyanin content in theextract is at least 10% by weight of the extract product.

C. Identification of Anthocyanin and Novel Anti-Inflammatory Compounds

The present section is directed towards providing a general teaching ofthe purification and identification of such compound(s).

In general, the plant extract may be prepared as described herein above,obtained from another method, or obtained from a commercial source. Theplant extract will comprise a mixture of flavonoid compounds some ofwhich will have COX-2 selective activity, others of which will haveCOX-1 selective activity, still others which will have a broad spectrumcyclooxygenase inhibitory activity and still others which will not haveany appreciable inhibitory activity of cyclooxygenase inhibition.

Upon demonstrating that a particular plant extract has ananti-inflammatory activity, using for example the assays describedherein below or other equivalent assays known to those of skill in theart for measuring COX activity, it will be possible to separate theindividual components of the fruit extract. Separation techniques arewell known to those of skill in the art. For example, those of skill inthe art may employ chromatography such as thin layer chromatography, gaschromatography, high performance liquid chromatography, paperchromatography, affinity chromatography, ion exchange chromatography,supercritical flow chromatography and the like to separate theindividual flavonoid components (See Freifelder, Physical BiochemistryApplications to Biochemistry and Molecular Biology, 2nd ed., Wm. Freemanand Co., New York, N.Y., 1982 for an overview of chromatographictechniques).

Partition chromatography is based on the theory that if two phases arein contact with one another, and if one or both phases constitute asolute, the solute will distribute itself between the two phases.Usually, partition chromatography employs a column that is filled with asorbent and a solvent. The solution containing the solute is layered ontop of the column. The solvent is then passed through the column,continuously, which permits movement of the solute through the columnmaterial. The solute can then be collected based on is movement rate.The two most common types of partition chromatograph are paperchromatograph and thin-layer chromatograph (TLC); together these arecalled adsorption chromatography. In both cases, the matrix contains abound liquid. Other examples of partition chromatography as gas-liquidand gel chromatography.

Paper chromatography is a variant of partition chromatography that isperformed on cellulose columns in the form of a paper sheet. Cellulosecontains a large amount of bound water even when extensively dried.Partitioning occurs between the bound water and the developing solvent.Frequently, the solvent used is water. Usually, very small volumes ofthe solution mixture to be separated is placed at top of the paper andallowed to dry. Capillarity draws the solvent through the paper,dissolves the sample, and moves the components in the direction of flow.Paper chromatograms may be developed for either ascending or descendingsolvent flow. Two dimensional separations are permitted by changing theaxis of migration 90° after the first run.

Thin layer chromatography (TLC) is very commonly used to separate lipidsand, therefore, is considered a preferred embodiment of the presentinvention. TLC has the advantages of paper chromatography, but allowsthe use of any substance that can be finely divided and formed into auniform layer. In TLC, the stationary phase is a layer of sorbent spreaduniformly over the surface of a glass or plastic plate. The plates areusually made by forming a slurry of sorbent that is poured onto thesurface of the gel after creating a well by placing tape at a selectedheight along the perimeter of the plate. After the sorbent dries, thetape is removed and the plate is treated just as paper in paperchromatography. The sample is applied and the plate is contacted with asolvent. Once the solvent has almost reached the end of the plate, theplate is removed and dried. Spots can then be identified byfluorescence, immunologic identification, counting of radioactivity, orby spraying varying reagents onto the surface to produce a color change.

TLC of anthocyanins from bilberry extracts is described by Petri et al.,(1994). This reference also describes additional spectrophotmetric andchromatographic techniques that can be used in the identification andcharacterization of anthocyanin agents.

In gas liquid chromatography (GLC), the mobile phase is a gas and thestationary phase is a liquid adsorbed either to the inner surface of atube or column or to a solid support. The liquid usually is applied as asolid dissolved in a volatile solvent such as ether. The sample, whichmay be any sample that can be volatized, is introduced as a liquid withan inert gas, such as helium, argon or nitrogen, and then heated. Thisgaseous mixture passes through the tubing. The vaporized compoundscontinually redistribute themselves between the gaseous mobile phase andthe liquid stationary phase, according to their partition coefficients.

The advantage of GLC is in the separation of small molecules.Sensitivity and speed are quite good, with speeds that approach 1000times that of standard liquid chromatography. By using a non-destructivedetector, GLC can be used preparatively to purify grams quantities ofmaterial.

Gel chromatography, or molecular sieve chromatography, is a special typeof partition chromatography that is based on molecular size. The theorybehind gel chromatography is that the column, which is prepared withtiny particles of an inert substance that contain small pores, separateslarger molecules from smaller molecules as they pass through or aroundthe pores, depending on their size. As long as the material of which theparticles are made does not adsorb the molecules, the sole factordetermining rate of flow is the size. Hence, molecules are eluted fromthe column in decreasing size, so long as the shape is relativelyconstant. Gel chromatography is unsurpassed for separating molecules ofdifferent size because separation is independent of all other factorssuch as pH, ionic strength, temperature, etc. There also is virtually noadsorption, less zone spreading and the elution volume is related in asimple matter to molecular weight.

The gel material for gel chromatography is a three-dimensional networkwhose structure is usually random. The gels consist of cross-linkedpolymers that are generally inert, do not bind or react with thematerial being analyzed, and are uncharged. The space filled within thegel is filled with liquid and this liquid occupies most of the gelvolume. Common gels are dextran, agarose and polyacrylamide; they areused for aqueous solution.

High Performance Liquid Chromatography (HPLC) is characterized by a veryrapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofminutes, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample need not be very great because the bandsare so narrow that there is very little dilution of the sample. HPLC setup with a photodiode array detection system has been used to studyflavonoids such as rutin and other quercetin glycosides, phloridzin, aswell as certain anthocyanins (Paganga and Rice-Evans, FEBS Left.401(1):78-82, 1997). A reverse phase-HPLC gradient procedure has beendescribed for the separation and quantitative estimation of 12anthocyanins (Petri et al., Acta Pharm. Hung. 64(4) 117-122, 1994).Quercetin compounds also may be identified using the HPLC techniquesdescribed by Laires et al., (Food Chem. Toxicol., 31(12) 989-994, 1993).It is contemplated that such methods may be adapted to the presentinvention in characterizing and identifying novel flavonoids.

Affinity Chromatography is a chromatographic procedure that relies onthe specific affinity between a substance to be isolated and a moleculethat it can specifically bind to. This is a receptor-ligand typeinteraction. The column material is synthesized by covalently couplingone of the binding partners to an insoluble matrix. The column materialis then able to specifically adsorb the substance from the solution.Elution occurs by changing the conditions to those in which binding willnot occur (alter pH, ionic strength, temperature, etc.).

The matrix should be a substance that itself does not adsorb moleculesto any significant extent and that has a broad range of chemical,physical and thermal stability. The ligand should be coupled in such away as to not affect its binding properties. The ligand should alsoprovide relatively tight binding. And it should be possible to elute thesubstance without destroying the sample or the ligand. One of the mostcommon forms of affinity chromatography is immunoaffinity chromatographywhich employs antibodies directed against the particular materials to bedetected.

The structure of anthocyanins separated using the above techniques canbe identified by generating mass spectra and NMR spectra as described bySaito et al., (Phytochemistry 41(6) 1613-1620,1996 and Phytochemistry43(6), 1365-1370, 1.996); Takeda et al., (Phytochemistry, 36(3)613-616,1994). Additional NMR techniques are described by Terahara etal. (BioSci. Biotech. Biochem., 58(7) 1324-1325, 1994); Nerdal et al.,(Acta Chem. Scand. 46(9) 872-876, 1992). Johansen et al.,(Phytochemistry30(12)4137-4141, 1991) describe various methods includingion-exchange resin, droplet-counter chromatography and gel filtrationfor the isolation of anthocyanins and the subsequent use of techniquessuch as chemical degradation, chromatography and spectroscopy,especially homo- and heteronuclear two-dimensional NMR techniques forthe characterization of the isolated anthocyanin compounds. It will beclear to those of skill in the art that any of the above describedtechniques can be used to isolate and further purify the fruit extractsdescribed herein to identify the individual compounds responsible forthe anti-inflammatory activity.

D. Assays to Test for Anti-Inflammatory Activity

In the present invention, it is described that anthocyanin-containingplant extracts have an anti-inflammatory activity. More particularly, itis demonstrated that such extracts inhibit COX-2 activity preferentiallyover COX-1 activity. As such these extracts provide an excellentalternative to the traditional NSAIDs in that they are selective forCOX-2. These inhibitory extracts are further advantageous over therecently developed, COX-2 specific “super aspirins” because theseextracts are natural extracts that have not been linked to increasedpropensity for heart attacks, strokes, and other adverse cardiovascularevents.

The concentration of any inhibitor that inhibits the enzyme to 50% ofits maximal activity is called IC₅₀ or I₅₀. The smaller the IC₅₀, thestronger or more potent the corresponding inhibitor is for the enzymeinhibition. Consequently, a smaller amount of inhibitor would berequired for anti-inflammatory and pain-relief supplement formulation ifthe compound can be absorbed, metabolized, transported to themalfunctional or diseased site.

Some materials, compounds, or plant concentrates may selectively inhibiteither COX-1 or COX-2 enzyme. This can be referred to as selectivity ofthe material. The selectivity can be numerically expressed by the ratioof I₅₀ (COX-1)/I₅₀ (COX-2). When the ratio is equal to 1, the inhibitorhas no selectivity for either of the isozymes; i.e. the inhibitor isequally inhibiting COX-1 and COX-2 enzymes. When the ratio is less than1, the inhibitor is more selective for COX-1 inhibition. When the ratiois more than 1, the inhibitor is more selective for COX-2 inhibition.For chronic anti-inflammatory and pain-relief drugs or supplements, theselectivity may play a key role in side effects. The side effects aremostly gastrointestinal (GI) bleeding caused by the inhibition of COX-1enzyme on the GI tract where prostaglandins have a normal function on GIlining.

The selectivity is an important issue in non-sterol anti-inflammatorydrugs (NSAIDs), because NSAIDs only have one active form that caninhibit constitutively expressed COX 1 enzyme in GI tract and cause GIbleeding, in addition to the expected action of absorption andtransportation to the inflammatory and pain sites. Though not yetproven, natural products, such as anthocyanin-containing plant extracts,may have an advantage because they have non-active and active forms andtherefore, may not cause side effects in GI tract. Different mechanismsof absorption, metabolism and transportation may exist. It is possiblethat the non-active form (glycosidic form with sugar) can be absorbed orpassed through the GI tract without inhibiting the COX-1 enzyme there.As a result, the amount of prostaglandins generated by COX 1 enzyme onthe GI tract is normal or high enough to maintain the GI lining. Afterthe absorption, the sugar moiety is cleaved and the active form(aglycone form, anthocyanidin) is transported to the site where COX 2enzyme is induced at great level, although COX 1 will be inhibited aswell (but to a lesser degree). The inhibition of both enzymes on thesite will be very effective in anti-inflammation and pain relief.

In certain aspects of the present invention, it will be necessary todetermine whether a particular plant extract or a component thereofpossesses an anti-inflammatory activity. Such an activity may bemeasured using anti-inflammatory assays well known to those of skill inthe art. The use of prostaglandin endoperoxide synthase-1 and -2isozymes will allow a facile determination of whether a particularextract has the appropriate activity. These assays determine the abilityof these enzymes to convert arachidonic acid to prostaglandins.Alternatively, an immunoassay method as described below may be used.

Reagents such as arachidonic acid and microsomal suspension of theCOX-1, and COX-2 enzymes are readily available to those of skill in theart (e.g., from Oxford Biomedical Research, Oxford, Mich., USA).

Accordingly, COX-2 inhibitory activity of a particular extract may bemeasured using a method including generally the steps of (a) obtaining aCOX-2 microsomal composition; (b) admixing the candidate extract withthe COX-2 microsomal composition; and (c) determining the ability of thecandidate extract to inhibit the COX-2 activity.

COX-2 activity may be measured by obtaining a microsomal membranepreparation of COX-2 e.g., (5-10 mg protein/ml in an appropriatebuffer). COX-2 assay is performed at 37° C. by monitoring the rate of O₂uptake as described (DeWitt et al., Am. J. Med. 95(2A)40S-44S, 1993;Arch. Biochem. Biophys. 306(1) 96-102; 1993). This assay basicallymeasures the conversion of arachidonic acid to prostaglandinendoperoxide-2. Thus, one unit of cyclooxygenase activity represents theoxygenation of 1 nmol of arachidonic acid/minute (DeWitt et al., 1993supra). Alternatively, the activity of COX-2 may be measured usingchromatography by determining the amount of the product of the COX-2enzyme using e.g., thin layer chromatography, gas chromatography, highperformance liquid chromatography and the like. Yet another way tomeasure the COX-2 activity would be to employ radio-labeling ofsubstrates and monitoring the amount of radio-labeled end-product(s) ofthe COX-2 reaction. A preferred way to measure the COX activity is anenzyme immunoassay as is described in greater detail in Example 4.Regardless of the method employed one of skill in the art will be ableto tabulate the end measurement as a cyclooxygenase activity e.g., O₂used/mg cyclooxygenase/min; mg product/mg cyclooxygenase/min; μCiradio-labeled product produced/mg cyclooxygenase/min; μCi radio-labeledarachidonate used/mg cyclooxygenase/min.

To identify a fruit extract as being capable of inhibiting COX-2, onewould measure or determine the COX-2 activity of the microsomalpreparation in the absence of the added candidate extract. One wouldthen add the candidate extract to the preparation and re-determine theactivity in the presence of the candidate extract. A candidate extractwhich reduces the amount of arachidonate oxygenated relative to thearachidonate oxygenation in its absence is indicative of a candidateextract with COX-2 inhibitory capability.

Control experiments can be conducted in which known inhibitors of COXactivity e.g., aspirin, ibuprofen, Celebrex™, naproxen and the like maybe used. By comparing the results of the fruit extract with that of theCOX-2 activity in the presence of these known inhibitors useful,relative activities also may be determined.

A significant decrease in arachidonate oxygenation, e.g., as measuredusing oxygen consumption with an O₂ electrode, chromatography techniques(quantitation of end-product by densitometry or liquid scintillationspectroscopy), are represented by a reduction in COX-2 activity levelsof at least about 20%-40%, and most preferably, by decreases of at leastabout 50%, with higher values, of course, being possible. Chromatographyassays that measure arachidonic acid metabolites and COX enzyme assaysthat measure prostaglandin formation are well known in the art and maybe conducted in vitro or in vivo.

Quantitative in vitro testing of the inhibitory properties of the fruitextract is not a requirement of the invention as it is generallyenvisioned that the fruit extracts that form the nutraceutical agents ofthe present invention will often be the same compounds that arenaturally found in the whole fruits. Of course, it should be understoodthat the anthocyanin and flavonoid compounds that form the COX-2inhibitory components of the fruit extracts described herein may furtherbe modified in vivo upon ingestion to produce the anti-inflammatorycompounds.

Similarly, in vivo testing is not a necessary requirement. However, oneof skill in the art may employ animal models of inflammation to test forthe in vivo activity of these compounds. For example, a rodent modelhaving an inflamed area may be used to test the anti-inflammatoryeffects of the COX-2 inhibitors that have been identified by assays suchas those described above. Such an animal model would be employed in anassay which would use, for example, at least two animals having asimilar inflammation, one of the animals would be contacted with thecandidate anti-inflammatory composition and the other animal would becontacted with a control or placebo composition which contains all thecomponents of the candidate composition with the notable exception thatit lacks the anti-inflammatory component. A reduction in inflammation ofthe animal contacted with the candidate composition as compared to theanimal contacted with the control or placebo composition would beindicative of the candidate composition having anti-inflammatoryactivity.

E. Formulations

The present invention provides a natural food supplement made fromextracts wherein the food supplement comprises an anti-inflammatoryactivity that is greater than the anti-inflammatory activity found inthe natural fruit. The present invention provides an extract that can bepresented in a powdered, liquid, or solid form. Specific formulationsare provided herein below in the Examples, the present section discussesthe forms and components of formulations that would be desirable andreadily produced given the teachings of the present invention.

The extract is likely a reconstitutable powder composition that, whenreconstituted with, for example, water, milk or some other similarliquid will provide a drink, which may be used to provide ananti-inflammatory activity to a subject in need thereof. The powderedcomposition and drink prepared therefrom are especially useful as anenterally administered component in a program of pain or inflammationmanagement which utilizes a number of carefully designed products invarious forms, i.e., in shake, soup, fruit drink, snack bar and othersolid forms such as tablets, gel caps, and the like, which can be mixedand matched over a period of pain management to provide more attractiveand, therefore, more effective support to a patient, particularly thosein extended care situations.

In addition to drinks, the extracts of the present invention may be usedin foodstuffs. Such extracts may be combined with any other foodstuff,for example, oils containing the extracts of this invention may be usedas cooking oil, frying oil, or salad oil and may be used in anyoil-based food, such as margarine, mayonnaise or peanut butter. Grainflour fortified with the compounds of this invention may be used infoodstuffs, such as baked goods, cereals, pastas and soups. Oilscontaining the extracts and novel anthocyanins extracted therefrom canbe emulsified and used in a variety of water-based foodstuffs, such asdrinks, including drink mixes as discussed above. Advantageously, suchfoodstuffs may be included in low fat, low cholesterol or otherwiserestricted dietary regimens.

A “nutraceutical” is any functional food that provides an additionalbenefit other than its nutritional benefit. This category may includenutritional drinks, diet drinks (e.g., Slimfast™, Boost™ and the like)as well as sports herbal and other fortified beverages. The presentinvention provides nutraceutical compositions that may be used as ananti-inflammatory agent. As such, it can be used to relieve anycondition that is mediated by the action of COX-2 including but notlimited to, arthritis, headache, allergic rash, inflammatory bowelsyndrome, joint pain, chronic fatigue, fibromyalgia and the like.

In addition to the purified extract, the nutraceutical or foodstuff alsomay contain a variety of other beneficial components including but notlimited to essential fatty acids, vitamins and minerals. Thesecomponents should be well known to those of skill in the art, however,without being bound to any particularly formulations or content thepresent section provides a brief discussion of components that couldform part of the food supplements of the present invention. Additionaldisclosure describing the contents and production of nutritionalsupplements may be found in e.g., U.S. Pat. No. 5,902,797; U.S. Pat. No.5,834,048; U.S. Pat. No. 5,817,350; U.S. Pat. No. 5,792,461; U.S. Pat.No. 5,707,657 and U.S. Pat. No. 5,656,312 (each incorporated herein byreference.) Essential fatty acids such as γ-linolenic acid (ω-3) andlinoleic acid (ω-6) may be added to the food supplements of the presentinvention. Research has shown that in animals other than humans, theratio of n-3 to n-6 fatty acids is more important even than absoluteamounts of the fatty acids. Boudreau M D, et al., “Lack of Dose Responseby Dietary n-3 Fatty Acids at a Constant Ratio of n-3 to n-6 Fatty Acidsin Suppressing Eicosanoid Biosynthesis from Arachidonic Acid,” Am. J.Clin. Nutr. 54:111-117 (1991). Essential fatty acids are involved incardiovascular health as well as in support of the immune system. Animbalance in these essential fatty acids can lead to poor cholesterolmetabolism. Additionally, the immune system function can becomeimpaired, leading to inflammation.

Both calcium and magnesium are involved in bone health, among otherfunctions. One possible effect of an imbalance between calcium andmagnesium is an imbalance in bone minerals that can affect bone growthand bone turnover (the breaking down and building-up of bone). Magnesiumis equally as important as calcium for bone health and reducing the riskof osteoporosis, which affects men as well as women (Purvis, J. R.,“Effect of Oral Magnesium Supplementation Factors on SelectedCardiovascular Risk Factors in Non-Insulin-Dependent Diabetics,”Archives of Family Medicine 3:503-508 (1994).

The minerals zinc and copper are both involved in cardiovascular health,and should be provided in a ratio of 5:1 zinc:copper. An imbalancebetween these two minerals can cause an antagonistic effect of zinc oncopper. This effect can interfere with the body's ability to use copperfor supporting cardiovascular health. Too much zinc relative to coppercan also interfere with the body's ability to manufacture SOD(superoxide dismutase), an important heart-protective enzyme. Also, aproper zinc:copper ratio is required to achieve a proper balance of HDL(high density lipoproteins) to LDL (low density lipoproteins). Zincintake in the typical American man's diet is only 33 to 75 percent ofRDA as such dietary supplements that include zinc are contemplated.

Selenium and iodide also have a ratio at which they function mosteffectively, which is the ratio of selenium to iodide of about 2:1.These minerals affect thyroid function, and therefore also have theresulting effects on metabolism caused by changes in thyroid function.Imbalanced thyroid function can put undue stress on the body that willresult in malabsorption of nutrients from food. This, in turn, canretard growth and development.

Pyridoxine, folate and cobalamin also have a ratio at which theyfunction most effectively in the prevention of vascular disorders. Theoptimal ratio of pyridoxine (vitamin B6) to folate to cobalamin (vitaminB 12) is about 100:4:1, respectively. These vitamins affectcardiovascular function through their abilities to reduce the levels ofthe potentially toxic amino acid homocysteine. This ratio recognizes theimbalanced and inadequate levels of these vitamins consumed byindividuals at risk of heart disease from their diet.

In addition, vitamin C, vitamin B1 (thiamin), and vitamin E also can beprovided. Vitamin C requirements are increased in smokers and cigarettesmoking is a major contributor to lung cancer. Vitamin B1 plays anessential role in energy transformation. Thiamin diphosphate (TDP) is acoenzyme necessary for the conversion of carbohydrates to energy. SinceU.S. men currently consume about 45% of their total calories fromcarbohydrates, vitamin B1 optimization in the diet is desirable.

Along with vitamin B6, vitamin B12 and folic acid supplementation helpmodulate blood levels of homocysteine and as such will be usefulcomponents in the dietary supplement formulations of the presentinvention. Vitamin D (calciferol) is essential for formation of theskeleton and for mineral homeostasis. Without vitamin D, the smallintestine cannot absorb adequate calcium regardless of how much calciumis available for absorption. Thus, vitamin D is indicated as a componentof a nutritional supplement to help build strong bones.

The role of manganese in driving metalloenzyme manganese-superoxidedismutase (Mn-SOD) has been clearly identified, along with a similarrole in other metalloenzyme systems (glutamine synthetase, arginase, andpyruvate carboxylase). Numerous enzyme systems have also been shown toundergo manganese activation, even though they are not manganesemetalloenzymes. The manganese-SOD connection may be of special clinicalimportance, since this form of the metalloenzyme appears to be the soleoperative form within the cell's mitochondrial membranes, and thus mayplay a unique role in protection of the mitochondria and assurance ofthe body's oxidative energy production system. The inclusion ofmanganese in a dietary supplement would be desirable.

Additional micronutrients that may be included in the supplementsinclude but are not limited to the vitamins such as vitamin A, vitaminC, vitamin E, riboflavin, niacin, niacinamide, pantothenic acid,pyridoxine, cobalamin, biotin, inositol, choline bitartrate, betaine,and vitamin K and minerals such as molybdenum, chromium and potassium.

Stress, exercise, and other conditions create free radicals in the body,which can cause damage to the body's components. To counter the freeradicals, the present invention may include the following antioxidantsin addition to vitamins C and E discussed above: citrus bioflavonoids,mixed carotenoids, green tea extract, and N-acetylcysteine.

In addition other flavorings and additives well known to those of skillin the art also may be added to the formulations to make them morepalatable. For example, formulations may contain ginger, boswellia,fruit flavoring, coloring, preservatives and the like.

When ingested in a solid form, the nutraceutical composition of theinvention may additionally contain a solid carrier such as a gelatin oran adjuvant. When administered in liquid form, a liquid carrier such aswater, petroleum, oils of animal or plant origin such as peanut oil,mineral oil, soybean oil, or sesame oil, or synthetic oils may be added.In some formulations, it may be desirable to use the cake that resultsfrom the extraction process used to obtain the anthocyanin-containingextract as some or all of the carrier. For example, cherry cake is aby-product of the cherry juice industry. Often these cakes containbeneficial components such as anthocyanins and bioflavonoids and whenused as the carrier, should increase the potency of the formulation. Thenutraceutical composition of the present invention may also containstabilizers, preservatives, buffers, antioxidants, or other additivesknown to those of skill in the art.

In a preferred embodiment, a dietary or nutritional supplement ornutraceutical is provided and contains from about 0.1% to about 99%,preferably from about. 30% to about 90% of an anthocyanin-containingextract. In this regard, a single dosage form (i.e., a single tablet,capsule, serving (whether liquid or solid)) contains from about 1 mg. toabout 500 mg. of total anthocyanin, preferably from about 5 mg. to about100 mg., more preferably from about 20 mg. to about 70 mg. of totalanthocyanin. In a preferred formulation, a tablet (a single dosage form)is provided that contains about 35 mg. of total anthocyanin. The phrase“total anthocyanin” refers to the total amount of anthocyanin present inthe single dosage form.

The extract obtained from an anthocyanin-containing plant is selectedfrom the group consisting of peonidin, cyanidin, pelargonidin,delphinidin, petunnidin, malvidin, kaempferol, hesperidin,gentiodelphin, platyconin, cinerarin, including their glycosidederivatives, and mixtures thereof. In one a preferred embodiment, theanthocyanins are selected from the group consisting of cyanidin,peonidin, malvidin, petunidin, delphinidin, their glycoside derivatives,and mixtures thereof. Avantageously, the nutritional supplement containsthe stable anthocyanin, which will be hydrolyzed in vivio to theaglycone form, anthocyanidin, to provide COX inhibition activity.

A preferred nutritional supplement contains a fruit extract, wherein thefruit extract is selected from the group consisting of an extract ofelderberry, tart cherry, bilberry, and mixtures thereof. Moreparticularly, the fruit extract comprises an extract of elderberry in anamount from about 2% to about 98% by weight of the fruit extract, anextract of tart cherry in an amount from about 1% to about 49% by weightof the fruit extract, and an extract of bilberry in an amount from about1% to about 49% by weight of the fruit extract. Preferably, the extractcomprises from about 90% to about 98% (more preferably about 96%) of anelderberry extract, from about 1% to about 5% (more preferably about 2%)of a cherry extract (preferably tart cherry), and from about 1% to about5% (more preferably about 2%) of a bilberry extract. In this preferrednutritional supplement, cyanidin-based anthocyanins comprise at leastabout 90% by weight of the total anthocyanins present in the elderberryextract. Preferably, cyanidin-based anthocyanins comprise about 95% andmore preferably about 96% by weight of the total anthocyanins present inthe elderberry extract. In this regard, the cyanidin is present as amixture of cyanidin-3-glucoside, cyanidin-3-sambunigrin,cyanidin-3,5-diglucoside, and cyanidin-3-samb-5-glucoside. Likewise,cyanidin-based anthocyanins comprise at least about 90% by weight of thetotal anthocyanins present in the tart cherry extract. Preferably,cyanidin-based anthocyanins comprise about 95% and more preferably about96% by weight of the total anthocyanins present in the tart cherryextract. In contrast to the elderberry, the cyanidin is present as amixture of cyanidin-3-glucosyl rutinoside, cyanidin-3-rutinoside,cyanidin-3-glucose, cyanidin-3-rutinoside-haxose,cyanidin-3-rutinoside-pentose, and cyanidin-3-rutinoside. Finally, thebilberry contains a mixture of malvidin, peonidin, cyanidin, petunidin,and delephinidin based anthocyanins. Each of these anthocyanins compriseabout 95% and more preferably about 96% by weight of the totalanthocyanins present in the bilberry extract. More particularly, themalvidin is present as malvidin-3-arabinoside, malvidin-3-glucoside,malvidin-3-galactoside. The peonidin is present as peonidin-3-lucoside,peonidin-3galactoside. The cyanidin, petunidin, and delphinidin arepresent as the 3-glucoside and 3-galactoside.

F. Combinations of Anthocyanins with Other Anti-Inflammatory Agents

The present invention in certain aspects describes the beneficial intakeof a food supplement having anti-inflammatory properties wherein thefood supplement comprises an extract having an anti-inflammatoryactivity greater than the anti-inflammatory activity found in thenatural fruit. Those of skill in the art should understand that such afood supplement may advantageously be combined with otheranti-inflammatory agents. Such additional anti-inflammatory agents will,of course, be secondary to the extracts of the present invention and maybe any commonly recognized anti-inflammatory agent or indeed may be onethat is identified by using the assay presented herein above.

Regardless of whether the additional anti-inflammatory agent is a knownanti-inflammatory or is identified using the present invention, thepresent invention will contemplate the use of various combinations thatmay be employed. Thus, where the fruit extract is “A” and the otheranti-inflammatory agent is “B” the combinations may be as follows:A/B/A   B/A/B   B/B/A A/A/B   A/B/B   B/A/A A/B/B/B B/A/B/BB/B/B/A     B/B/A/B   A/A/B/B     A/B/A/B   A/B/B/A B/B/A/AB/A/B/A     B/A/A/B   A/A/A/B     B/A/A/A   A/B/A/A A/A/B/A

The extract and the additional anti-inflammatory agent may be contactedwith or exposed to a cell either in vivo or in vitro to inhibit theCOX-2 activity of the cell. The terms “contacted” and “exposed,” whenapplied to a cell are used herein to describe the process by which anextract and a second anti-inflammatory agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve a beneficial effect, both agents may be delivered to a cell in acombined amount effective to inhibit COX-2 activity, decreaseinflammation, and decrease the production of the inflammation causingprostaglandins or other such effect that will decrease the inflammatoryresponse in a cell or an individual subject in which the cell islocated.

Anti-inflammatory agents are well known to those of skill in the art andinclude agents such as salicylic acid derivatives (e.g. aspirin)paraminophenol derivatives (e.g. acetaminaphen) indole and indene aceticacids (indomethacin, sulindac and etodalac) heteroaryl acetic acids(tolmetin diclofenac and ketorolac, aryl propionic acid derivatives(ibuprofen, naproxen, keopren, fenopren, oxaprozine), anthranilic acids(mefenamic acid, meclofenamic acid) enolic acids (piroxicam, tenoxicam,phenylbutazone and oxyphenthatrazone). These and other anti-inflammatoryagents are well known to those of skill in the art and no additionaldescription of these agents need be provided.

G. Combinations with Other Active Therapeutic Agents

According to another embodiment of the present invention, theanthocyanins and/or novel compounds derived from natural sources may becombined with other therapeutic agents. It is contemplated that suchcombinations may, in some cases, provide synergistic effects.

It is intended that the anthocyanins and/or novel compounds can becombined with any suitable therapeutic agents. In a preferredembodiment, however, a nutritional supplement is provided that containsnatural active ingredients. As a result, the preferred active agents arethose obtained or derived from natural sources and can include varioussuitable extracts, for example herbal extracts. The PDR® for HerbalMedicines, the entire contents of which is incorporated herein byreference, provides a suitable listing of the types of herbal and/orplant extracts that may be suitable for combining with the anthocyaninsof the present invention.

In one embodiment, a joint health agent is provided with one or moreanthocyanin-containing extract for use in improving the joint health ina mammal. In this embodiment, the joint health agent is selected fromthe group consisting of glucosamine, chondroitan, ginger, boswellia,tumeric, curcumin, fever few, bromelain, and salts, derivatives, andmixtures thereof. In yet another embodiment, a dietary supplementincludes at least one prostate health agent that is effective tomaintain or improve normal prostrate function with an effective amountof an anthocyanin-enriched plant extract having an anti-inflammatoryactivity greater than the anti-inflammatory activity found in the plant.In this embodiment, the prostrate health agent is selected from thegroup consisting of saw palmetto, pumpkin seed, nettle root, and salts,derivatives and mixtures thereof. Another embodiment includes at leastone gamma-linolenic acid (GLA) agent that is effective to maintain orimprove a woman's general well being during her menstrual cycle incombination with an effective amount of an anthocyanin-enriched plantextract. In this embodiment, the GLA agent is obtained from a sourceselected from the group consisting of evening primrose, borage, blackcurrant, chasteberry, ginger, and salts, derivatives, and mixturesthereof.

H. Methods of Use of the Novel Formulations

The formulations described above may be used to prevent, reduce, oreliminate the symptoms and conditions associated with pain andinflammation. In addition, these formulations when used to treatinflammation and pain are desirable because they have minimalgastrointestinal side effects. These formulations may also be used in ananti-oxidant capacity to reduce or inhibit the oxidation of a materialthat results from the exposure to free radicals. Furthermore, theseformulations may include the use of anthocyanins with other therapeuticagents that are useful to provide a synergistic result that does notoccur with the therapeutic agents alone.

Pain is often defined as the sensory and emotional experience associatedwith actual or potential tissue damage. Pain can be influenced byphysical, mental, biochemical, physiological, social, cultural andemotional factors. Pain is typically categorized as two types; acute orchronic. Acute pain is usually characterized by relatively short, sharppain. When acute pain persists beyond the expected time required forhealing, it is considered chronic pain. (C. Thomas, ed., Taber'sCyclopedic Medical Dictionary, F. A. Davis Company, 1993).

Pain is perceived when the nerves that receive and transmit painfulstimuli, nociceptors, are stimulated. Nociceptors are located in skin,bones, joints, muscles, and internal organs. The various types ofnociceptors sense sharp blows, heat, pressure, temperature, chemicalchanges or inflammation. They transmit the pain signal to other nervesthat send messages to the spinal cord and brain. (Cashman J. N., Themechanisms of action of NSAIDs in analgesia, Drugs, 1996; 52:13-23.)

The mechanism of pain first involves the stimulation of the nociceptors.When the nociceptrors are stimulated, the release of substance P, aneuropeptide that modulates cellular responses and increases the painmessage, is increased. Substance P can stimulate the release ofbradykinin, a neuropeptide that propagates slow pain. Histamine is alsoreleased, which evokes pain. Prostaglandins are released by damagedtissue during an inflammation response. Prostaglandin E series (PGEs),prostaglandin I₂(PGI₂), and leukotriene B₄(LTB₄) amplify pain byincreasing the sensitivity of the nociceptors to the effects of othermediators or mechanical stimuli. This is called hyperalgesia. (Gilman A,Rall T, Nies A, Taylor P eds, Goodman and Gilman's The PharmacologicalBasis of Therapeutics, New York, Pergamon Press, 1990.

The mechanism of inflammation typically involves 4 main symptoms:redness, warmth, edema (swelling), and pain. When tissue is damaged bymechanical, chemical, biological, or invading organisms, the first thingto happen is that mast cells release histamine. The histamine stimulatesdilation of the blood vessels. The increase in blood volume to the areacauses redness and the sensation of warmth. Kinins are released, whichpotentiate the vasodilation. The vasodilation causes plasma thatcontains mediators of acute inflammation (complement, C-reactiveprotein, antibodies, neutrophils, eosinophils, basophils, monocytes, andlymphocytes) to leak into the surrounding tissue. This causes the tissueto look swollen. The loss of plasma from the blood causes blood tobecome more viscous. The blood platelets and leukocytes start to sticktogether and clump. Platelet aggregation causes the platelets to releaseserotonin, which participates in the formation of pain. The damagedtissue and cell membranes cause an influx of calcium into the cells,which activates the enzyme phospholipase A₂ Phospholipase A₂ acts on thephospholipids to release arachidonic acid and produce thepro-inflammatory agent called platelet-activating factor. Neutrophilscontaining lipoxygenase create chemotactic compounds from arachidonicacid. This provokes the release of cytokinins that potently activateinducible cyclo-oxygenase 2 (COX-2) and inducible nitric oxide synthase(NOS). (Gilman A, Rall T, Nies A, Taylor P eds, Goodman and Gilman's ThePharmacological Basis of Therapeutics, New York, Pergamon Press, 1990.Robak J, Gryglewski R J, Bioactivity of flavonoids, Pol J Pharmacol,1996, 48:555-564.

The enzyme cyclo-oxygenase (COX) acts on arachidonic acid to generateprostaglandin (PG) G₂, and PGH₂. PGG₂ and PGH₂ are not stable and areconverted into prostacyclin (PGI₂) by PGI₂ synthase, thromboxane (TXA₂)by TXA₂ synthase, and stable prostaglandins PGD₂, PGE₂, PGF₂ ^(α) . NOSforms nitric oxide and free radicals. Free radicals increase membranepermeability and provide a chemotactic signal for specialized cells, ie,neutrophil polynuclears, macrophages, and lymphocytes. Also, the enzymelipoxygenase can act on arachidonic acid to generate leukotrienes from5-HETE. The end result is inflammation and pain. (Cashman J. N., Themechanisms of action of NSAIDs in analgesia, Drugs, 1996; 52:13-23;Robak J, Gryglewski R J, Bioactivity of flavonoids, Pol J Pharmacol,1996, 48:555-564).

Pain can be caused by a variety of conditions, and certain classes ofdrugs that relieve pain are unsuitable for specific types of pain.Therefore, determining the cause of the pain is important in determiningthe most effective treatment. For example, neuropathic pain caused bynerve damage, or complex regional pain syndromes are effectively treatedwith antidepressants, anticonvulsants, or α2-agonists, but not opioids(i.e. morphine). Crampy intestinal or constipation-induced pains arebest treated with antispasmodic drugs; opioids are not effective.Opioids are most effective at treating acute pain syndromes. Arthritiscan be treated with nonsteriodal anti-inflammatory drugs (NSAIDs),whereas peptic ulcer pain is not helped with NSAIDs. Finally,psychological, such as ‘phantom pain’ or spiritual pain can not betreated with analgesics. (Wilder-Smith CH, Pain treatment in multimorbidpatients, the older population and other high-risk groups. The clinicalchallenge of reducing toxicity, Drug Saf., 1998,18:457-472.)

Currently, there are five main categories of drugs available to treatpain. These include 1) analgesics (e.g. acetaminophen), 2) salicylates(e.g. aspirin) and other NSAIDs (e.g. ibuprofen and naproxen), 3) opoiddrugs (e.g. codeine and morphine), 4) corticosteroids, and 5) adjuvantagents (e.g. antidepressants, anticonvulsants) (Aronson, M. D.,Nonsteroidal anti-inflammatory drugs, traditional opoids, and tramadol;Clin Ther. 1997; 19:420-432. (Aronson M D, Nonsteroidalanti-inflammatory drugs, traditional opioids, and tramadol: contrastingtherapies for the treatment of chronic pain, Clin Ther.,1997,19:420-432, discussion 367-428.)

The formulations described herein include at least oneanthocyanin-containing compound from a natural source that may be usedto alleviate or reduce pain in a mammal. In one embodiment of theinvention, the compound is an extract obtained from ananthocyanin-containing plant selected from the group consisting of sweetcherry, tart (sour) cherry, acerola cherry, plum, bilberry, blackberry,black currant, red current, chokeberry, blueberry, strawberry,cranberry, boysenberry, grapes, red raspberry, black raspberry,elderberry, loganberry, barberry, pomegranate, red cabbage, blue andpurple potatoes, purple carrot, black beans, black soybeans, hibiscus,echinacea purpea, and mixtures thereof. A preferred group ofanthocyanin-containing plants include strawberry, acerloca cherry, plum,cranberry, grapes, pomegranate, red cabbage, blue and purple potatoes,purple carrot, and echinacea purpea. A more preferred group ofanthocyanin-containing plants include sweet cherries, blueberries, redrasberry, blackberry, loganberry, black beans, black soybeans,hibiscous, and barberry. The most preferred anthocyanin-containingplants include elderberry, chokeberry, tart cherry, bilberry, blackraspberries, and boysenberry. In another embodiment, elderberry ispreferred as the natural source of the anthocyanin-containing compound.

The extract from the anthocyanin-containing plant is provided in anamount that is effective to alleviate or reduce pain. The extract istypically included in a unit dosage form. In general, the extractcomprises at least about 1% by weight of the unit dosage form. Dependingon the extract used and the diluent or excipient that is appropriate forthat extract, the extract may comprise up to about 99% by weight of theunit dosage form. Generally, an extract containing at least about 4% byweight of anthocyanins will be effective to alleviate or reduce pain. Inanother embodiment, an extract that is substantially free ofanthocyanidins will be effective to reduce or alleviate pain. As usedherein, “substantially free” means that the extract does not containanthocyanidins that are formed when anthocyanins are hydrolyzed.However, an extract that is substantially free of anthocyanidins mayinclude small amounts of anthocyanidins that normally occur in thenatural source or small amounts that are formed by a concentratingprocess, excluding any hydrolyzation processes.

The anthocyanin-containing extract can be used to alleviate or reduceacute pain or chronic pain. In yet another embodiment, theanthocyanin-containing extract is used to alleviate or reduce one ofmore symptoms of pain. Pain symptoms that may be alleviated or reducedinclude, but are not limited to, headache, joint pain, muscular pain,dysmenorrhea, inflammation, and combinations thereof. Formulations thatinclude an effective amount of an anthocyanin-containing extract arewell suited for treating or controlling inflammation related diseasessuch as arthritis and gout. These anthocyanin-containing extracts can beused for inhibiting the cyclooxygenase or prostaglandin H synthaseenzymes. In one particular embodiment, the cyclooxygenase orprostaglandin H synthase enzymes are inhibited by providing ananthocyanin selected from the group consisting of cyanidin-3-glucoside,cyanidin-3,5-glucoside, cyanidin-3-sambubioside,cyanidin-3-sambubioside-5-glucoside, and mixtures thereof isolated fromthe fruit of elderberry to inhibit the enzymes.

When dual inhibitor NSAIDs (NSAIDs that inhibit both the COX-1 and COX-2enzymes) are used long-term to relieve pain and/or inflammation, therisk of developing gastrointestinal complications is moderate to high.Examples of gastrointestinal complications include ulcers, perforationof the stomach or intestines, gastric outlet obtruction and bleeding. Incontrast, formulations that include an anthocyanin-containing extractcan be used to reduce or alleviate pain and/or inflammation with minimalgastrointestinal side effects. In one embodiment, a method ofalleviating or reducing pain or its symptoms in a mammal is providedwherein the method does not produce significant erosions in the gastriclining. In another embodiment, the method does not exhibit a significantpropensity to induce gastric or intestinal ulceration.

I. EXAMPLES

The following examples are included to demonstrate a preferredembodiment of the invention. However, those of skill in the art should,in light of the present disclosure, appreciate that many changes can bemade in the specific embodiment disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

Example 1

100 kilograms of cherries were pressed using a bag press and the juicewas collected. The collected juice was filtered at a temperature lessthan about 38° C. through an ultrafiltration unit having a 200,000molecular weight cutoff. The UF unit was operated so that the retentatecontained less than 0.5% by weight solids.

Thereafter, the permeate from the UF unit was subjected to reverseosmosis using a membranes having a 4,000 molecular weight cut off. Thereverse osmosis step continues until the retentate contains about 1% orless by weight solids.

The retentate is collected in a tank and concentrated to at least 20% byweight solids by a vacuum evaporator at a temperature of less than about52° C. to avoid degradation of the concentrated flavonoids.

The concentrate is combined with maltodextrin and spray dried with theoutlet temperature of the spray drier maintained at a temperature lessthan about 27° C.

The retained pulp from the bag press was collected, dried, and milled.

Example 2

A batch consisting of 38.8 kilograms of tart cherries was pressed in abag press to produce 19.3 kilograms of juice and 18.6 kilograms of cake.The juice, which had a pH of 3.3 was pumped to an ultrafiltrationmembrane at a flow rate between 1770 and 1950 g/min, a pressure of 10bar, and a temperature ranging from initial 29° C. at the start of thefiltration to 18° F. at the end of the filtration. A difiltrate flow wasinitiated and continued until the dissolved solids in the permeate wereabout 0.2% by weight.

The ultrafiltration membrane was a PVDF polymeric membrane having arated 100,000 molecular weight (Daltons) cut off. A suitable membranecan be obtained from PCI Membrane Systems under the tradename FP.

At the end of the ultrafiltration, 53.7 kilograms of permeate containing5% by weight solids and 3.49 kilograms of retentate containing 0.3% byweight solids was collected. The permeate was then subjected tonanofiltration/reverse osmosis at a feed pressure of 40 bar and a flowrate ranging initially from 1290 g/min to finally 1380 g/min attemperature of 24° C. at the start of the process and a temperature of41° F. at the completion of the process. A difiltrate of 72.4 kilogramsof water was used.

A polyethersulphone membrane having a 4,000 molecular weight (Daltons)cut off was used. A suitable membrane can be obtained from PCI MembraneSystems under the tradename ES404.

Upon completion of this step, 6.4 kilograms of retentate was collectedand it contained 1% by weight solids. 117 kilograms of permeatecontaining 2% by weight solids was recovered.

To produce a powder, the retentate was combined and mixed with 79 gramsof maltodextrin M100 and the resulting product was introduced into aspray drier with an inlet temperature of about 140° C. and an outlettemperature of about 90° C. to produce about 105 grams of powder.

Example 3 Comparison of COX-2 and COX-1 Inhibitory Activity of FruitExtracts

The present example describes an enzyme assay method using an oxygenmonitoring system to monitor the COX inhibitory activity of fruitextracts. In this assay the changes of concentration of dissolved oxygenare constantly monitored by an oxygen electrode in a Dissolved OxygenMeasuring System (Instech, Plymouth Meeting, Pa.). The output isrecorded by a linear Recorder (Fisher Scientific, Pittsburgh, Pa.).

Each day a fresh potassium chloride solution (15 g/100 ml distilledwater) was made and the electrode was set up according to manufacturersinstructions. The chamber is kept at 37° C.

A prostaglandin assay kit was used and the assay set up in a mannersimilar to that described for the COX-1 assay. Briefly, 50 μl phenol wasadded to 20 ml 100 mM Tris buffer, warmed to 37° C. for 1 minute(working buffer). To a tube of hematin 0.9 ml of the working buffer wasadded. 50 μl 0.1NaOH was added to an arachidonic acid vial and vortexed.0.43 ml water was added and the solution mixed again. Samples ofextracts were weighed and dissolved in the working buffer to a finalconcentration of 0.1 g/ml. Buffer, samples or diluted samples are usedin the enzyme assays directly.

The enzyme assay is performed according to the manufacturer'sinstruction, which should be well known to those of skill in the art.Briefly, 600 μl of working buffer were drawn into the chamber from theoverflow outlet with the injection valve close, and the main outletconnected to a syringe in the right orientation. The stir bar was set aspeed of 3 k/min. At 1 minute intervals, 5 ul enzyme, 15 ul hematinsolution, 6 ul buffer or sample or diluted sample and 8 ul arachidonicacid solution was injected. The oxygen concentration changes weretransformed into mV and recorded. When the arachidonic acid was added,the consumption of oxygen or the decrease of oxygen concentration wasapparent.

There were clear trends in selectivity among the samples tested asindicated by the tables given below. TABLE 3 COX-1 and COX-2 IC50 valuesand Specific Activity Sample COX-1 IC50 COX-2 IC50 Specific Activity*Aspirin  1/20,000 1/10,000 0.5 Montmorency Tart  1/20,000 Not availableNA Cherry Primer Balaton Tart 1/5,000 1/23,000 4.6 Cherry Prime MilneTart cherry 1/2,500 Not available NA Artemis Blueberry >>1/1,000 1/10,000 NA Artemis 1/2,000 1/15,000 7.5 Chokeberry Artemis Elderberry1/3,000 1/23,000 10.1  Nutrilite Acerola  1/20,000 Not available NACherry Celebrex 1/4,000 1/28,000 7   Quercetin standard 1/3,500 1/22,0006.3 Artemis Bilberry  1/12,000 1/15,000  1.25*The higher this number the more selective the extract is for COX-2 asopposed to COX-1.

In addition the COX-2 potency was monitored in relation to Celebrex, awell known COX-2 specific inhibitor. TABLE 4 COX-2 Potency in relativeto Celebrex The method described above was used to determine the COX-2inhibition of various inhibitor candidates and compared to Celebrex, aknown COX-2 inhibitor. Potency compared to Sample COX-2 Celebrex ArtemisChokeberry 1/15,000 54% Artemis Elderberry 1/23,000 82% ArtemisBlueberry 1/10,000 36% Balaton Tart Cherry Prime 1/23,000 82% QuercetinStandard 1/22,000 78% Celebrex 1/28,000 100% 

The data clearly shows the selectivity of the Artemis dark berry samplestowards COX-2.

Example 4

A presently preferred method of determining whether an inhibitorcandidate inhibits either COX-1 or COX-2 is described below. In general,for each inhibitor candidate, six different concentrations are used forboth COX 1 and COX 2 enzyme reactions. The content of PG-f2α fromstandards, or enzyme reactions are quantitated by an immunoassay. Theamount of PG-f2α in standards is used to make a standard curve (opticaldensity vs. concentration) and the standard curve is used to calculatethe amount of PG-f2α in each enzyme reaction (regression) for thesamples. Then, the content of PG-f2α the six different reactionconcentrations of the same inhibitor candidate is used to make a samplecurve. Finally, the concentration of the inhibitor candidate thatinhibits the enzyme to 50% of its maximal activity (with no inhibitors),or 150 is obtained from the sample curve. To keep the resultsconsistent, one inhibitor candidate or drug is used for each set ofexperiments as a positive control.

COX-1 and COX-2 enzymes were obtained from Dr. Daniel Tai at theUniversity of Kentucky. They were prepared as follows: COX-1 enzyme wasextracted from human platelet concentrate obtained from the CentralKentucky Blood Center. The platelet suspension was centrifuged at1,000×g for 10 min. The pellet was washed with the same volume ofphosphate-buffer saline and the suspension was again centrifuged. Theplatelets were suspended in 5 volumes of 50 mM Tris-HCl buffer, pH 7.5,and subjected to sonication for 3×20 sec at 4° C. The suspension wascentrifuged at 5,000×g for 10 min. The supernatant was furthercentrifuged at 100,000×g for 60 min. The pellet (microsomes) wassuspended in 5 ml of 50 mM Tris-HCl buffer, pH 7.5 and stored in 200 μlaliquots at −80° C. This fraction was used as a source of COX-1 enzyme.

Recombinant human COX-2 enzyme was obtained from insect cells (Sf9)infected with recombinant baculovirus carrying COX-2 cDNA. Briefly, Sf9cells (1×10⁷) were seeded in 75 cm² tissue culture flask in 20 ml ofcomplete TNF-FH medium. Cells were allowed to attach for 1 hour. Themedium was removed; 4 ml of Grace's medium containing recombinant virusat a multiplicity of about 10 was added. The cells were allowed to growcontinuously for 72 hours. Cells were collected by centrifugation at500×g for 10 min. The cells were then suspended in 1 ml of 50 mMTris-HCl, pH 7.5 buffer and sonicated for 3×10 sec at 0° C. Thehomogenate was briefly spun at 5,000×g for 5 sec to remove cell debris.The supernatant was then stored in 200 μl aliquots at −80° C. Thisfraction was used as a source of COX-2 enzyme.

The following buffers were prepared: 1. Coating Buffer:  0.1MNaHCO₃/Na₂CO₃, pH 9.5 2. enzyme immunoassay  0.1M KH₂PO₄/K₂HPO₄, pH7.5(“EIA”) Buffer: containing 0.9% NaCl and 0.1% bovine serum albumin(ELISA or RIA grade) 3. Antibody Stabilizing EIA buffer plus sucroseBuffer: (5 g per 100 ml) 4. Washing Buffer: 0.01M KH₂PO₄/K₂HPO₄, pH7.5containing 0.05% Tween 20 5. Enzyme reaction buffer and 50 mM Tris-HCl,pH sample dilution buffer: 7.5 6. PBS (phosphate buffer 10 mMKH₂PO₄/K₂HPO₄, pH7.5 saline) containing 0.9% NaCl 7. Protein A solution1 mg/ml in PBS

The wells for the immunoassay were coated by (a) adding 100 μl protein Asolution to 19.9 ml coating buffer, mixing well, and pouring into adispensing tray; (b) pipetting 200 μl of the above to each well (rinsemany times before delivering to wells); (c) storing the plates at roomtemperature for 4-5 h or 37° C. for 2-3 h or 4° C. overnight; (d)pipetting 100 μl EIA buffer to each well to block the unfilled sites,shaking and incubating at room temperature for 2 h or at 4° C.overnight. The plates can be stored at 4° C. for an indefinite time ifthe wells are supplied with water.

The following solutions were prepared: 1. Arachidonic acid:   1 mg/ml inethanol 2. Isoproterenol: 2.5 mg/ml, prepare immediately before use 3.Hemoglobin: 3.2 mg/ml, prepare immediately before use 4. SnCl₂  50 mg/mlin ethanol 5. HCl 1 N 6. K-blue substrate buffer: Neogen, Lexington, KY7. COX-1 enzyme (as described 30 times dilution, use 5 μl per above):assay 8. COX-2 enzyme (as described 5 times dilution, use 5 μl perabove) assay 9. PGF-2α-antibody (Dr. Tai) 5,000× dilution, use 50 μl perwell 10. PGF-2α-HRP (Dr. Tai) 2,000× dilution, use 100 μl per well

The following PGF-2α standards were prepared:

-   -   A. 1 μg/ml    -   B. 20 μl A (1 μg/ml) plus 980 μl EIA buffer 1,000 pg/50 μl    -   C. 200 μl B plus 1.8 ml EIA buffer 100 pg/50 μl

D. 200 μl C plus 1.8 nl EIA buffer 10 pg/50 μl TABLE 5 Standard (pg/50μl/well) B C D Buffer (ml) 0 1.0 5 0.5 0.5 10 1.0 20 0.2 0.8 50 0.5 0.5100 1.0 200 0.2 0.8 500 0.5 0.5 1000 1.0

Where the inhibitor candidates were extracts of anthocyanin-containingplants, the anthocyanins were extracted, concentrated, and hydrolyzed toprovide the aglycone form, e.g., the anthocyanidin. Similarly, where theinhibitor candidates were commercial extracts, the extracts werehydrolyzed to provide the aglycone form. In each case, it was theaglycone form of the anthocyanins that were tested. The hydrolyzedanthocyanins were then dissolved in 0.1% HCl in methanol for testing.

PGF-2α is a prostaglandin and an enzyme reaction is conducted todetermine whether the inhibitor candidate effectively inhibits eitherCOX-1 or COX-2 (depending on the enzyme being used). PGF-2α is theindirect stable prostaglandin reduced from the prostaglandin productsformed by the enzyme reaction.

The enzyme reaction procedure was conducted as follows: (a) 385 μl of abuffer (50 mM Tris-HCl, pH 7.5) was prepared and mixed at roomtemperature with 50 μl of isoproterenol, 10 μl of hemoglobin, 511 ofSnCl₂, and 5 μl of enzyme (depending on the enzyme to be tested, e.g.COX-1 or COX-2 described above); (b) 455 μl of the mixture (a) was addedto tubes that contained 40 μl of the inhibitor candidate, mix; (c) add 5μl of the arachidonic acid solution to each tube, mix, and incubate at37° C. for 5 min.; (d) stop the reaction by adding 30 μl of the 1 N HCland mixing; (e) neutralize by adding 30 μl of 1 M Tris-base.

The enzyme immunoassay procedure is a method to measure the amount ofPGF-2α generated by the enzyme reaction. The enzyme immunoassay wasconducted in the following manner: (a) shake out all the liquid in thewell and blot on the paper towel; (b) wash w/200 μl washing buffer 2times, shake and blot; (c) add 50 μl of the PGF-2α-antibody; (d) add 50μl STD (PGF-2α) or the enzyme reaction product from above (diluted 50×with EIA buffer); (e) add 100 μl PGF-2α-HRP (in EIA buffer); (f) shakeand allow the plate to stay at room temperature for 1 hour; (g) washwells three times by repeating step (b); (h) add 100 μl substrate bufferto each well; (i) incubate at room temperature for 3 to 30 minutesdepending on the color development; (j) turn on the computer and 96-wellbioassay reader, and follow the operation instructions (MolecularDevices, V_(max) kinetic microplate reader); (k) add 30 μl 1 N HCl toterminate the reaction; (I) read at 420 nm.; (m) save the data.

The data is analyzed by: (a) copying the data to a spreadsheet, (b)making a standard curve according to the standard PGF-2α immunoassayresults; (c) finding the PGF-2α amount in all sample enzyme reactionsusing regression from the standard curve; (d) drawing a curve for eachsample; and (e) determining I₅₀. In accordance with this procedure,several inhibitor candidates were evaluated and the results in set forthin Table 6, below. TABLE 6 COX Inhibition Final Vol. Concentration(hydrolyzed liquid samples) Sample After of total Concentration I₅₀ (COXWeight purification anthocyanidins of cyanidin Cyanidin I₅₀ (COX 1) 2)I₅₀ (COX 1)/ Samples (g) (ml) (mg/ml) (mg/ml) (%) (ug) (ug) I₅₀ (COX 2)A 1.33 2.1 1.53 0.93 61 13.2 10.7 1.2 B 0.15 2.0 1.68 0.74 44 19.2 21.40.9 C 0.15 2.0 2.92 1.36 47 8.7 8.7 1.0 D 0.2028 1.0 0.20 0.20 100 34.417.5 2.0 E 0.15213 1.0 0.17 0.17 100 31.5 15.8 2.0 F 0.30784 1.0 0.0570.046 80 32.8 23.5 1.4 Aspirin 100 100 1 Ibuprofen 8 2 4 Celebrex 7 1 7Vioxx 10 0.5 20A is a tablet that contains 40% bilberry with 7% anthocyaninB is a commercial extract from Nutritech containing 25% bilberryC is a commercial extract from Nutritech containing 25% bilberry from aproduction lot different from sample BD is a tablet that contains 39.17% of elderberry that has 15%anthocyanins.E is a tablet that contains 39.17% of elderberry that has 20%anthocyanins.F is a tablet that contains 11.76% of elderberry having 15% anthocyaninsand 19% bilberry having 7.2% anthoycanins.Elderberry has 98% cyanidin and its glycosides.Bilberry has 23% cyanidin and its glycosides.

From the above, it appears that the higher cyanidin content increasesthe potency and selectivity.

Example 5

In accordance with the procedure described in Example 4 above, a numberof inhibitor candidates were evaluated. Table 7 presents the results.TABLE 7 I₅₀ I₅₀ Content Extract COX-1 COX-2 Selectivity actives Bilberry(from Artemis)* 29 22 1.3 10.0% Rubini (from Artemis)* 75 57 1.2  7.2%Elderberry proto. 18 14 1.3 21.0% Bilberry (from Iprona)* 41 34 1.2 2.3% Chockberry (from Iprona)* 117 97 1.2 13.6% Elderberry (fromIprona)* 47 35 1.3 17.0% Bilberry (from Nutratech)* 14 10 1.4 10.6% TartCherry 01-01a* 250 200 1.3 3.48% Elderberry 004-03*(23% 25 19 1.315.06%  CRR) Elderberry 004-04*(0% CRR) 18 14 1.3 20.15%  Pomegranateextract powder 120 80 1.5 n/a Tumeric Extract 250 150 1.7 BoswelliaSerrata 200 150 1.3 Panax gotogingseng 200 250 0.8 Ginger 200 100 2Green Tea Extract Powder 70 60 1.2 Green Tea Polyphenols 110 100 1.1Note:1. Total anthocyanins are quantified as cyanidin-3-glucoside for allfruit samples.2. Content of actives is calculated based on test results and percentageof active compounds. E.g. . 100% of anthocyanins in elderberry,chockberry and tart cherry are actives cyanidin glycosides.*means that COX inhibitory activities are tested from hydrolyzed and XADcolumn purified fruits.

In accordance with the above results, a food supplement for thetreatment of inflammation preferably provides a COX-1 inhibition to aCOX-2 inhibition of at least 1, and preferably greater than 1.3. As aresult, the food supplement will provide a selective inhibition ofCOX-2.

Example 6 Specific Formulations

The present example provides formulations containing one or more fruitextracts from anthocyanin-containing plants for use as anti-inflammatoryagents. Of course, these are merely exemplary formulations and those ofskill in the art will understand that these formulations may be alteredaccording to particular specifications and yet still remain equivalentto the formulations of the present invention. TABLE 8 Anti-inflammatoryFormulation 1 2 Unit Component formula 1 unit formula % formula ActiveIngredients: Elderberry Extract (min.  100 mg   50 mg 11.277% 7%anthocyanin) Chokeberry Extract min.  100 mg   50 mg 11.277% 10%anthocyanin Tart Cherry Extract 5.00 mg 2.50 mg 0.564% Excipient: RicePowder 675.00 mg  337.5 mg  76.121% Magnesium Stearate 4.50 mg 2.25 mg0.507% Silicone Dioxide 2.25 mg 1.13 mg 0.254%

TABLE 9 Anti-inflammatory Formulation 2 2 Unit 1 unit Component formulaformula % formula Active Ingredients: Elderberry Extract (min. 13%  100mg   50 mg 11.855% anthocyanin) Chokeberry Extract (min. 10%  100 mg  50 mg 11.855% anthocyanin) Tart Cherry Extract 5.00 mg 2.50 mg 0.593%Other Anti-inflammatory Herbal Extract: Boswellia serrata extract 600.00mg  300.00 mg  71.132 (min. 65% boswellic acids) Excipient: Rice Powder25.00 mg  12.50 mg  2.964% Magnesium Stearate 9.00 mg 4.50 mg 1.067%Silicone Dioxide 4.50 mg 2.25 mg 0.533%

TABLE 10 Anti-inflammatory Formulation 3 2 Unit 1 unit Component formulaformula % formula Active Ingredients: Elderberry Extract (min. 13%   100mg    50 mg 5.905% anthocyanin) Chokeberry Extract (min. 10%   100 mg   50 mg 5.905% anthocyanin) Tart Cherry Extract  5.00 mg  2.50 mg0.295% Other Anti-inflammatory Herbal Extract: Boswellia serrata extract600.00 mg 300.00 mg 35.430% (min. 65% boswellic acids) Ginger Extract(min. 5% 500.00 mg 125.00 mg 29.525% gingerols) Excipient: Rice Powder375.00  93.75 22.143% Magnesium Stearate  9.00 mg  2.25 mg 0.531%Silicone Dioxide  4.50 mg  1.13 mg 0.266%

TABLE 11 Pain/Inflammation relief formulation Mg of anthocyanin providedin a single unit dosage 1 unit Component % formula formula ElderberryExtract (min.  33% 32 mg 7% anthocyanin) Bilberry Extract 3.5%  2 mgTart Cherry Extract 3.5%  .4 mg Excipients  60% N/A

Example 7

Thirty-seven men and women were selected to participate in an open-labeltrial to determine the pain relief capability of a berry blend dietarysupplement. The dietary supplement used in this trial was comprised ofelderberry, bilberry, and tart cherry, as described in Table 11. Thesupplement contained about 34 mg of anthocyanins total in a singletablet. The participants were randomized into One Tablet or Two Tabletsgroups. Subjects in the One Tablet group were instructed to take one,and subjects in the Two Tablets group were instructed to take two, BerryBlend tablets to relieve episodes of mild pain as needed. Subjectsrecorded their pain on a 10-cm visual analogue scale (VAS; 0=no pain,10=worst possible pain) before as well as 30 to 45 min after takingBerry Blend. If pain relief was inadequate subjects were free to taketheir regular pain medication once they had recorded their “After” painintensity on the VAS.

The predefined success criterion was greater pain relief in the TwoTablets than in the One Tablet group, as measured by group mean percentpain reduction in a one-tailed t-test with alpha set at 0.05. Subjectsin the One and Two Tablets group had equivalent baseline VAS-rated pain[means±standard deviation for one and two tablet groups respectively:4.11±1.38 and 4.35±1.71, t (35)=0.47, p=0.64, two-tailed]. Afterconsumption of the test products both groups reported pain reduction(FIG. 2), with a trend towards the Two Tablets group showing greatermean percent pain reduction than the One Tablet group [One Tablet:38.7%±30.1; Two Tablets: 52.4%±29.9, t (35)=1.52, p=0.07, one-tailed](FIG. 3).

In this trial, Headaches were the most common pain type, representingover half of all reports (FIG. 4). Next most common was joint/muscular,a combined category where no attempt was made to differentiate bydiagnosis or history of disease; about one third of reports were in thiscategory. Dysmenorrhea represented about 7-12% of pain reports and afinal “other” category, for cases not captured by the precedingcategories, represented about 5% of reports. There were not enoughsubjects in each of these categories to allow analysis of differentialpain relief by pain category.

Subjects were also asked to estimate how long the product took beforemeaningful pain relief was felt (on a catergorical scale of 15 min toover 60 min), and to give an “approval” rating of how they thought theproduct worked for them (from Poor to Excellent). Two tablets tooksignificantly less time to provide pain relief than did one tablet[about 45 min vs. about 60 min, t (35)-1.7, p=0.04, one-tailedcomparison of categorical time-to-relief] (FIG. 5). The overall“approval” rating was slightly higher for the double dose than thesingle dose [t (35)=1.5, p=0.07, one-tailed], and for both this wassomewhere between “fair” and “good.”

Example 8

The same participants in the trial described in Example 7 were testedone week apart with ibuprofen 400 mg and Berry Blend two tabletsfollowed by the same challenge, a 500-ml water-based sucrose solution.In this test, the participants drank a solution containing a measuredamount of sucrose (table sugar), and a 5-h collection of their urine wasassayed for sucrose concentrations. When gastric tissue is intact,sucrose is digested in the stomach and can not be found intact in theurine. When there are ulcers or minute erosions in the gastric liningwhich can precede ulcers, sucrose leaks through the gastric tissuebefore it can reach the stomach. The sucrose is then eventually excretedintact in the urine. As such, urinary sucrose concentrations serve as anindex of gastric damage such as erosions in the gastric lining andgastric or intestinal ulceration.

Subjects had less sucrose permeability following Berry Blend thanfollowing ibuprofen [t (5)=2.9, p=0.04, two-tailed Wilcoxon matchedpairs] (FIG. 6), suggesting Berry Blend is gentler to the gastric mucosathan ibuprofen.

Example 9

Two dietary supplements were analyzed qualitatively by LC-MS and PDA.The first supplement contained elderberry powder. The results of thisanalysis are shown in FIG. 7. The second supplement is a combination ofelderberry, bilberry and tart cherry as described in Table 11. Theresults of this analysis are shown in FIG. 8. The following is a key forthe compounds shown in FIGS. 7 and 8:

-   -   Compound a=Cyanidin-3-sambubioside-5-glucoside (M⁺=743)    -   Compound b=Cyanidin-3-sambubioside (M⁺=581)    -   Compound c=Cyanidin-3-glucoside (M⁺=449)    -   Compound d=Cyanidin (M⁺=287)    -   Compound e=Rutin (M+1=611)    -   Compound f=Quercetin (M+1=303)    -   Compound g=Chlorogenic acid (M+1=335)    -   Compounds u/A=unspecified anthocyanins    -   Compounds u=unidentified flavones and polyphenolic compounds

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

1-44. (canceled)
 45. A food supplement having anti-inflammatoryproperties wherein the food supplement comprises: (a) ananthocyanin-enriched extract having an anti-inflammatory activitygreater than the anti-inflammatory activity found in the natural fruit,wherein the extract is obtained from two or more plants selected fromthe group consisting of acerola, bilberry, black currant, black beans,black raspberry, black soybeans, blackberry, blueberry, boysenberry,carrott (purple), chokeberry, cranberry, echinacea, elderberry, grapes,hibiscus, loganberry, plum, pomegranate, potatoes (black, blue andpurple), red cabbage, red currant, red raspberry strawberry, sweetcherry, tart (sour) cherry and mixtures thereof, and wherein the extractprovides a greater cyclooxygenase 2 (COX-2) inhibitory activity thancyclooxygenase 1 (COX-1) inhibitory activity; and (b) a pharmaceuticallyacceptable diluent or excipient. 46-56. (canceled)
 57. The foodsupplement of claim 45 wherein the extract is obtained from elderberryand bilberry.
 58. The food supplement of claim 45 wherein the extract isobtained from elderberry and blueberry.
 59. The food supplement of claim45 wherein the extract provides at least about 25 mg of totalanthocyanins per a unit dose wherein 95% of the antyocyanins arecyanidin glycosides.